JP6415322B2 - T cell receptor recognizing HLA-A1- or HLA-CW7-restricted MAGE - Google Patents

Description

CROSS REFERENCE TO RELATED APPLICATIONS This application claims the priority of US Application No. 61 / 535,086, filed Sep. 15, 2011, which is hereby incorporated by reference in its entirety.

INCORPORATION BY REFERENCE TO ELECTRONIC SUBMITTED CHARACTERISTICS A computer-readable nucleotide / amino acid sequence listing co-submitted with the present specification and identified as follows is hereby incorporated by reference in its entirety: 2012 One file dated August 22, 1710922ST25.TXT, 52,162 bytes ASCII (text) file.

  Adoptive cell therapy (ACT) involves the transfer of reactive T cells to patients (including the transfer of tumor reactive T cells to cancer patients). Adoptive cell therapy using T cells targeting human leukocyte antigen (HLA) -A2 restricted T cell epitopes has been successful in causing tumor regression in some patients. However, patients without HLA-A2 expression cannot be treated with T cells that target HLA-A2 restricted T cell epitopes. Such restrictions are an obstacle to the wide application of adoptive cell therapy. Accordingly, there is a need to improve immunological compositions and methods of cancer treatment.

SUMMARY OF THE INVENTION The present invention has antigen specificity for a) melanoma antigen family A (MAGE A) -3 in relation to HLA-A1, or b) MAGE-A12 in relation to HLA-Cw7. An isolated or purified T cell receptor (TCR) is provided. The invention further provides related polypeptides and proteins and related nucleic acids, recombinant expression vectors, host cells and cell populations. Further provided by the invention are antibodies related to the TCR of the invention, or antigen-binding portions thereof, and pharmaceutical compositions.

  Further provided by the present invention are methods for detecting the presence of cancer in a host and methods for treating or preventing cancer in a host. The method of the present invention for detecting the presence of cancer in a host comprises the steps of (i) using a sample comprising cancer cells as a TCR, polypeptide, protein, nucleic acid, recombinant expression vector, host cell of the present invention as described herein. Forming a complex by contacting either the host cell population, or the antibody or antigen-binding portion thereof, and (ii) detecting the complex. Detection of such a complex indicates the presence of cancer in the host.

  The methods of the present invention for treating or preventing cancer in a host can include any of the TCRs, polypeptides or proteins described herein, any of the TCRs, polypeptides, or proteins described herein. Any host cell or host cell comprising any nucleic acid or recombinant expression vector comprising a nucleotide sequence encoding or a recombinant vector encoding any of the TCRs, polypeptides or proteins described herein. Administration of the population in an amount effective to treat or prevent cancer in the host.

Brief description of some of the drawings
FIG. 1A shows untransduced (UT) cells (black bar graph) or anti-MAGE-A3 TCR A10 (SEQ ID NO: 46) (unshaded bar graph) in response to co-culture with various tumor cell lines. ) Or anti-MAGE-A3 TCR 13-18 (SEQ ID NO: 48) (grey bar graph) is a bar graph showing the secretion (pg / ml) of interferon (IFN) -γ in cells transduced. FIG. 1B shows HLA-A1 + / MAGE-A3 + fresh tumor FrTu2767 (black bar), FrTu3178 (gray bar), FrTu2823 (unshaded bar) or FrTu3068 (shaded bar), or HLA-A ^* 0201 + / MART-1 + fresh tumor FrTu2851 (horizontal bar graph) or FrTu3242 (vertical bar graph) or UT cells or anti-MAGE-A3 TCR A10 (SEQ ID NO: 46) or anti-MART -1 is a bar graph showing IFN-γ secretion (pg / ml) of cells transduced with TCR DMF5. The checkered bar graph represents cells that are not co-cultured with tumor cells. Figures 2A and 2B are truncated human low affinity nerve growth factor receptor (NGFR) (black bar graph), anti-MAGE-A12 TCR 502 (SEQ ID NO: 47) in response to co-culture with various tumor cell lines. (Unshaded bar graph), or from first (FIG. 2A) and second (FIG. 2B) donors transduced with a control contract encoding anti-MAGE-A12 TCR FM8 (SEQ ID NO: 49) (grey bar graph). It is a bar graph which shows the secretion (pg / ml) of the cell IFN-gamma. FIG. 3A shows the cumulative percentage of normal white population that expresses HLA-A1 (the unshaded part of the bar graph), HLA-A2 (the gray part of the bar graph), and / or HLA-Cw7 (the black part of the bar graph). Is a bar graph illustrating FIGS. 3B and 3C show HLA-A2 and NY-ESO-1 (hatched portion of the bar graph), HLA-A1 and MAGE-A3 (unshaded portion of the bar graph); HLA-A2, MAGE-A3 and MAGE -A12 (gray part of the bar graph); and / or human melanoma (FIG. 3B) and synovial cell sarcoma (FIG. 3C) patient population as would be expected to express HLA-Cw7 and MAGE-A12 (black part of the bar graph) It is a bar graph illustrating the accumulation rate. FIG. 4 shows peptides of MAGE-A12 (VRIGHTLYIL; SEQ ID NO: 4), MAGE-A2 (VPISHLYIL; SEQ ID NO: 50), MAGE-A3 (DPIGHLYIF; SEQ ID NO: 51), MAGE-A6 (DPIGVYIF; SEQ ID NO: 52), Or NGFR (black bar graph), anti-MAGE-A12 TCR 502 (SEQ ID NO: responsive to co-culture with HLA-Cw ^* 0701 and HLA-Cw ^* 0702 target cells pulsed with a control peptide (EDGCPAAEK; SEQ ID NO: 53) 47) (bar graph without shading) or bar graph showing IFN-γ secretion (pg / ml) of cells transduced with anti-MAGE-A12 TCR FM8 (SEQ ID NO: 49) (grey bar graph). Figures 5A-5D show untransduced PBMC (black circles) or anti-MAGE-A12 TCR 502 (SEQ ID NO: 47) (▼), anti-MAGE at the effector to target ratio (E: T). -A12 TCR FM8 (SEQ ID NO: 49) (diamond), anti-MAGE-A3 TCR A10 (SEQ ID NO: 46) (square), anti-MAGE-A3 TCR 13-18 (SEQ ID NO: 48) (▲) or anti-MAGE-A3 TCR FIG. 6 is a line graph showing the lysis rate of 397 mel (A), 624 mel (B), 2984 mel (C), and 2661 RCC (D) cells by PBMC transduced with 112-120 (white circles). One representative result from two independent experiments is shown. FIG. 6A shows untransduced cells (control) (striped bar graph) or anti-MAGE-A3 TCR A10 (SEQ ID NO: 46) (shaded) in response to co-culture with various tumor cell lines. (Bar graph) or anti-MAGE-A3 TCR 13-18 (SEQ ID NO: 48) (checkered bar graph) is a bar graph showing IFN-γ secretion (pg / ml) of cells transduced. Two representative results of 3 independent experiments evaluating the response of T cells transduced with these TCRs are shown. FIG. 6B shows anti-MAGE-A3 TCR A10 (SEQ ID NO: 46), anti-MAGE-A3 TCR 13-18 (SEQ ID NO: 48), anti-MAGE-A12 TCR 502 (SEQ ID NO: 47), or anti-MAGE-A12 TCR FM8. FIG. 5 is a bar graph showing the predicted relative copy number of vector DNA measured for cells transduced with (SEQ ID NO: 49). FIG. FIG. 6C shows anti-MAGE-A3 TCR A10 (SEQ ID NO: 46) (circle) or anti-MAGE-A3 TCR 13 in response to co-culture with target cells incubated with various concentrations of MAGE-A3 168-176 peptide. FIG. 5 is a line graph showing the amount of IFN gamma secreted by cells transduced with −18 (SEQ ID NO: 48) (square). FIG. 6D shows anti-MAGE-A12 TCR 502 (SEQ ID NO: 47) (shaded bar graph) or anti-MAGE-A12 TCR FM8 (SEQ ID NO: 49) (checkered pattern) in response to co-culture with various tumor cell lines. Is a bar graph showing the secretion (pg / ml) of IFN-γ of the transduced cells. Two representative results of 3 independent experiments evaluating the response of T cells transduced with these TCRs are shown. FIG. 6E shows anti-MAGE-A12 TCR 502 (SEQ ID NO: 47) (circle) or anti-MAGE-A12 TCR in response to co-culture with target cells incubated with various concentrations of MAGE-A12: 170-178 peptide. It is a line graph which shows the amount of IFN-gamma secreted by the cell transduced with FM8 (sequence number 49) (square). FIG. 6F shows untransfected cells (control) (striped bar graph) or anti-MAGE-A12 TCR 502 (SEQ ID NO: 47) (shaded) in response to co-culture with various tumor cell lines. Bar graph) or IFN-γ secretion (pg / ml) of cells transduced with anti-MAGE-A12 TCR FM8 (SEQ ID NO: 49) (checkered bar graph). Two representative results of 3 independent experiments evaluating the response of T cells transduced with these TCRs are shown. FIG. 7A shows untransduced cells (control) (striped bar graph) or anti-MAGE-A3 TCR A10 (SEQ ID NO: 46) (shaded) in response to co-culture with various fresh uncultured tumors. Is a bar graph showing IFN-γ secretion (pg / ml) of cells transduced with anti-MAGE-A3 TCR 13-18 (checkered bar graph). Representative results of one out of three independent experiments evaluating the response of T cells transduced with these TCRs are shown. FIG. 7B shows untransduced cells (control) (striped bar graph) or anti-MAGE-A12 TCR 502 (SEQ ID NO: 47) (shaded) in response to co-culture with various fresh uncultured tumors. Bar graph) or IMG-γ secretion (pg / ml) of cells transduced with anti-MAGE-A12 TCR FM8 (SEQ ID NO: 49) (checkered bar graph). Representative results of one out of three independent experiments evaluating the response of T cells transduced with these TCRs are shown. FIG. 8A is untransduced overnight co-cultured with target cells transfected with any of MAGE-A3, A1, A2, A4, A6, A9, A10 or A12 in addition to HLA-A ^* 01 Transduced with cells (control) (striped bar graph), or anti-MAGE-A3 TCR A10 (SEQ ID NO: 46) (shaded bar graph) or anti-MAGE-A3 TCR 13-18 (checkered bar graph) It is a bar graph which shows the secretion (pg / ml) of IFN-gamma of a cell. FIG. 8B shows transduced overnight co-cultured with target cells transfected with any of MAGE-A3, A1, A2, A4, A6, A9, A10 or A12 in addition to HLA-C ^* 07 ^: 02. Cells (control) (striped bar graph), or anti-MAGE-A12 TCR 502 (SEQ ID NO: 47) (shaded bar graph) or anti-MAGE-A12 TCR FM8 (SEQ ID NO: 49) (checkered bar graph) ) Is a bar graph showing IFN-γ secretion (pg / ml) of the cells transduced in (1). FIG. 8C shows transduced overnight co-cultured with target cells transfected with any of MAGE-A3, A1, A2, A4, A6, A9, A10 or A12 in addition to HLA-C ^* 07 ^: 01. Cells (control) (striped bar graph), or anti-MAGE-A12 TCR 502 (SEQ ID NO: 47) (shaded bar graph) or anti-MAGE-A12 TCR FM8 (SEQ ID NO: 49) (checkered bar graph) ) Is a bar graph showing IFN-γ secretion (pg / ml) of the cells transduced in (1). 9A, 9B show untransduced (control) CD8 + cells (FIG. 9A) or CD4 + cells (FIG. 9B) (striped bar graph) or anti-MAGE-A3 TCR co-cultured with various tumor targets. FIG. 5 is a bar graph showing IFN-gamma secretion of cells transduced with A10 (SEQ ID NO: 46) (shaded bar graph) or anti-MAGE-A3 TCR 13-18 (checkered bar graph). A representative result of one out of two independent experiments is shown. FIG. 9C shows non-transduced CD8 + cells (control) (striped bar graph) or anti-MAGE-A12 TCR 502 (SEQ ID NO: 47) (shaded bar graph) co-cultured with various tumor targets. Alternatively, it is a bar graph showing IFN-gamma secretion of cells transduced with anti-MAGE-A12 TCR FM8 (SEQ ID NO: 49) (checkered bar graph). A representative result of one out of two independent experiments is shown.

DETAILED DESCRIPTION OF THE INVENTION Embodiments of the invention include: a) melanoma antigen family A (MAGE A) -3 (also known as MAGE-3) in connection with HLA-A1, or b) HLA-Cw7. Provides a T cell receptor (TCR) having antigen specificity for MAGE-A12 (also known as MAGE-12).

  MAGE-A3 and MAGE-A12 also include MAGE-A1, MAGE-A2, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9, MAGE-A10 and MAGE-A11. It is a member of the MAGE-A family of 12 types of homologous proteins. MAGE-A protein is a cancer / testis antigen (CTA) that is expressed only in tumor cells and non-MHC-expressing embryonic cells of testis and placenta. MAGE-A protein is melanoma, breast cancer, leukemia, thyroid cancer, gastric cancer, pancreatic cancer, liver cancer (eg, hepatocellular carcinoma), lung cancer (eg, non-small cell lung cancer), ovarian cancer, multiple myeloma, esophageal cancer. Expressed in a variety of human cancers, including kidney cancer, head cancer (eg, squamous cell carcinoma), cervical cancer (eg, squamous cell carcinoma), prostate cancer, and urothelial cancer It is not limited.

  The TCR of the present invention provides many advantages, including when used for adoptive cell transfer. For example, by targeting a) MAGE-A3 presented in the context of HLA-A1, or b) MAGE-A12 presented in the context of HLA-Cw7, the TCR of the present invention is It makes it possible to treat patients that cannot be treated with a TCR that targets the MAGE antigen presented in the context of an HLA molecule (eg HLA-A2). Since HLA-A1 and HLA-Cw7 are widespread alleles, the TCR of the present invention advantageously significantly increases the patient population that can be treated. In addition, without being bound to a particular theory, since MAGE-A3 and / or MAGE-A12 are expressed in multiple types of cancer cells, the TCR of the present invention advantageously has multiple types of It is believed to provide the ability to destroy cancer cells and thus treat or prevent multiple types of cancer. In addition, without being bound by any particular theory, the MAGE-A protein is a cancer / testis antigen that is expressed only in tumor cells and non-MHC-expressing embryonic cells of the testis and placenta. Are advantageously targeted at the destruction of cancer cells while at the same time minimizing or eliminating the destruction of normal, non-cancerous cells, for example by minimizing or eliminating toxicity This is considered to be reduced.

  The phrase “antigen specificity” as used herein indicates that the TCR can specifically bind to MAGE-A3 or MAGE-A12 with high affinity and recognize them immunologically. means. Low concentrations of MAGE-A3 peptide or MAGE-A12 peptide, respectively (eg, about 0.05 ng / ml-about 5 ng / ml, 0.05 ng / ml, 0.1 ng / ml, 0.5 ng / ml, 1 ng / ml) Or 5 ng / ml) TCR-expressing T cells at least about 200 pg / ml or more (eg, 200 pg / ml) when cocultured with pulsed antigen negative HLA-A1 + target cells or HLA-Cw7 + target cells, respectively. ml or more, 300 pg / ml or more, 400 pg / ml or more, 500 pg / ml or more, 600 pg / ml or more, 700 pg / ml or more, 1000 pg / ml or more, 5,000 pg / ml or more, 7,000 pg / ml or more, 10,000 pg For example, TCR is MAGE-A3 or It may be considered as having an "antigen specificity" to MAGE-A12. Alternatively, or in addition, TCR-expressing T cells are pulsed with low concentrations of MAGE-A3 peptide or MAGE-A12 peptide, respectively, and antigen negative HLA-A1 + target cells or HLA-Cw7 + target cells, respectively. When T cells expressing TCR secrete IFN-γ at least twice the IFN-γ background level of untransduced PBL when co-cultured, the TCR is in MAGE-A3 or MAGE-A12. In contrast, it can be considered to have “antigen specificity”. The TCR of the present invention also secretes IFN-γ when co-cultured with antigen-negative HLA-A1 + target cells or HLA-Cw7 + target cells pulsed with higher concentrations of MAGE-A3 peptide or MAGE-A12 peptide, respectively. Can do.

  Embodiments of the present invention provide a TCR having antigen specificity for any MAGE-A3 protein, polypeptide or peptide. The TCR of the present invention may have antigen specificity for a MAGE-A3 protein comprising, consisting of, or consisting essentially of SEQ ID NO: 1. In a preferred embodiment of the invention, the TCR has antigen specificity for the MAGE-A3 168-176 peptide comprising, consisting of, or consisting essentially of EVDPIGHLY (SEQ ID NO: 2).

The TCR of the present invention can recognize MAGE-A3 depending on human leukocyte antigen (HLA) -A1-. “Depending on HLA-A1-”, as used herein, causes an immune response when a TCR binds to a MAGE-A3 cancer antigen in the context of an HLA-A1 molecule. Means. The TCR of the present invention can recognize MAGE-A3 presented by HLA-A1 molecules and can bind to HLA-A1 molecules in addition to MAGE-A3. Exemplary HLA-A1 molecules include those encoded by the HLA-A ^* 0101, HLA-A ^* 0102 and / or HLA-A ^* 0103 alleles and in connection with this molecule the TCR of the present invention. Recognizes MAGE-A3.

  Embodiments of the present invention provide a TCR having antigen specificity for any MAGE-A12 protein, polypeptide or peptide. The TCR of the present invention may have antigen specificity for the MAGE-A12 protein comprising, consisting of, or consisting essentially of SEQ ID NO: 3. In a preferred embodiment of the invention, the TCR has antigen specificity for the MAGE-A12 170-178 peptide comprising, consisting of, or consisting essentially of VRGHLYIL (SEQ ID NO: 4).

The TCR of the present invention can recognize MAGE-A12 depending on HLA-Cw7. “Depending on HLA-Cw7-” as used herein, causes an immune response when a TCR binds to a MAGE-A12 cancer antigen in the context of an HLA-Cw7 molecule. Means. The TCR of the present invention can recognize MAGE-A12 presented by HLA-Cw7 molecules and can bind to HLA-Cw7 molecules in addition to MAGE-A12. Exemplary HLA-Cw7 molecules include those encoded by the HLA-Cw ^* 0701 and / or HLA-Cw ^* 0702 alleles, in which the TCR of the invention recognizes MAGE-A12 To do.

  The present invention includes two polypeptides (ie, the TCR alpha (α) chain, the TCR beta (β) chain, the TCR gamma (γ) chain, the TCR delta (δ) chain, or combinations thereof (ie, A TCR comprising a polypeptide chain). Such TCR polypeptide chains are known in the art. If the TCR has antigen specificity for a) MAGE-A3 in the context of HLA-A1, or b) MAGE-A12 in the context of HLA-Cw7, the polypeptide of the TCR of the invention is Any amino acid sequence may be included.

  In an embodiment of the invention, the TCR comprises two polypeptide chains, each of which comprises a variable region comprising TCR complementarity determining regions (CDR) 1, CDR2, and CDR3. In an embodiment of the invention, the TCR has antigen specificity for MAGE-A3 168-176 and comprises the amino acid sequence of SEQ ID NO: 5 or 16 (a CDR1 of the α chain), the amino acid of SEQ ID NO: 6 or 17 A first polypeptide chain comprising a CDR2 comprising a sequence (a CDR2 of an α chain) and a CDR3 comprising an amino acid sequence of SEQ ID NO: 7 or 18 (a CDR3 of an α chain), and an amino acid sequence of SEQ ID NO: 8 or 19 A second comprising a CDR1 comprising a CDR1 comprising a amino acid sequence of SEQ ID NO: 9 or 20 (a CDR2 comprising a β chain) and a CDR3 comprising a amino acid sequence comprising SEQ ID NO: 10 or 21 (a CDR3 comprising a β chain) Contains a polypeptide chain. In other embodiments of the invention, the TCR has antigen specificity for MAGE-A12 170-178 and comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 26 or 36 (a CDR1 of the α chain), SEQ ID NO: 27 or 37 A first polypeptide chain comprising a CDR2 comprising an amino acid sequence (a CDR2 of an α chain) and a CDR3 comprising an amino acid sequence of SEQ ID NO: 28 or 38 (a CDR3 of an α chain), and an amino acid sequence of SEQ ID NO: 29 or 39 A second comprising a CDR1 comprising a CDR1 (β-chain CDR1), a CDR2 comprising the amino acid sequence of SEQ ID NO: 30 or 40 (a CDR2 of β-chain), and a CDR3 comprising the amino acid sequence of SEQ ID NO: 31 or 41 (a CDR3 of the β chain) Contains a polypeptide chain. In this regard, the TCR of the present invention is any one of SEQ ID NOs: 5-7, 8-10, 16-18, 19-21, 26-28, 29-31, 36-38, and 39-41. Any one or more of amino acid sequences selected from the group consisting of the above can be included. Preferably, the TCR comprises the amino acid sequence of SEQ ID NOs: 5-10, 16-21, 26-31, or 36-41. More preferably, the TCR comprises the amino acid sequence of SEQ ID NOs: 5-10 or 26-31.

  Alternatively, or in addition, the TCR may comprise the amino acid sequence of the variable region of the TCR that includes the CDRs described above. In this regard, a TCR having antigen specificity for MAGE-A3 168-176 is SEQ ID NO: 11 or 22 (α chain variable region), or 12 or 23 (β chain variable region), both SEQ ID NOs: 11 and 12. Or both amino acid sequences of SEQ ID NOs: 22 and 23. In other embodiments of the invention, the TCR has antigen specificity for MAGE-A12 170-178 and is SEQ ID NO: 32 or 42 (α chain variable region), or 33 or 43 (β chain variable region). ), Both amino acid sequences of SEQ ID NOs: 32 and 33, or both SEQ ID NOs: 42 and 43. Preferably, the TCR of the present invention comprises the amino acid sequences of both SEQ ID NOs: 11 and 12, or SEQ ID NOs: 32 and 33.

  Alternatively, or in addition, the TCR may comprise a TCR alpha chain and a TCR beta chain. Each of the α chain and β chain of the TCR of the present invention may independently contain any amino acid sequence. Preferably, the α chain comprises the variable region of the α chain described above. In this regard, the TCR of the present invention having antigen specificity for MAGE-A3 168-176 may comprise the amino acid sequence of SEQ ID NO: 13 or 24, and the TCR of the present invention having antigen specificity for MAGE-A12 170-178 is Or the amino acid sequence of SEQ ID NO: 34 or 44. This type of TCR of the present invention can be paired with any β chain of the TCR. Preferably, the β chain of the TCR of the present invention comprises the variable region of the β chain described above. In this regard, the TCR of the present invention having antigen specificity for MAGE-A3 168-176 may comprise the amino acid sequence of SEQ ID NO: 14 or 25, and the TCR of the present invention having antigen specificity for MAGE-A12 170-178 is Or the amino acid sequence of SEQ ID NO: 35 or 45. Thus, the TCR of the present invention comprises SEQ ID NOs: 13, 14, 24, 25, 34, 35, 44 or 45, both SEQ ID NOs: 13 and 14, both SEQ ID NOs: 24 and 25, both SEQ ID NOs: 34 and 35, Alternatively, it may comprise both amino acid sequences of SEQ ID NOs: 44 and 45. Preferably, the TCR of the present invention comprises the amino acid sequences of both SEQ ID NOs: 13 and 14, or SEQ ID NOs: 34 and 35.

  Further provided by the present invention is a polypeptide comprising a functional portion of any of the TCRs described herein. The term “polypeptide”, as used herein, refers to a single chain of amino acids, including oligopeptides, connected by one or more peptide bonds.

  For a polypeptide of the invention, if the functional moiety specifically binds to MAGE-A3 or MAGE-A12, the functional moiety can be any moiety that includes the contiguous amino acids of the TCR that it forms part of. The term “functional moiety” when used in reference to a TCR refers to any portion or fragment of a TCR of the present invention, which portion or fragment is the biological activity of the TCR (parent TCR) of which it is a part. Hold. The functional moiety may be, for example, the ability to specifically bind to MAGE-A2 (eg, dependent on HLA-A1) or MAGE-A12 (eg, dependent on HLA-Cw7), or the parent TCR It contains a portion of the TCR that retains the ability to detect, treat or prevent cancer to a close, similar or higher degree. With respect to the parent TCR, the functional portion may comprise, for example, about 10%, 25%, 30%, 50%, 68%, 80%, 90%, 95% or more of the parent TCR.

  A functional moiety can include additional amino acids at the amino terminus or carboxy terminus of the moiety, or at both termini, and such additional amino acids are not found in the amino acid sequence of the parent TCR. Desirably, the additional amino acid has a biological function of the functional moiety (eg, specifically binding to MAGE-A3 or MAGE-A12; and / or the ability to detect cancer and treat or prevent cancer, etc. Does not interfere with possession). More desirably, the additional amino acid increases the biological activity as compared to the biological activity of the parent TCR.

  The polypeptide is any one of the α chain and β chain of the TCR of the present invention, such as a functional part including one or more of CDR1, CDR2 and CDR3 of the variable region of the α chain and / or β chain of the TCR of the present invention. Both functional parts can be included. In this regard, the polypeptide is SEQ ID NO: 5, 16, 26 or 36 (α chain CDR1), 6, 17, 27 or 37 (α chain CDR2), 7, 18, 28 or 38 (α chain CDR3). , 8, 19, 29 or 39 (β-chain CDR1), 9, 20, 30 or 40 (β-chain CDR2), 10, 21, 31 or 41 (β-chain CDR3), or a combination thereof A functional part comprising a sequence may be included. Preferably, the polypeptide of the present invention comprises SEQ ID NOs: 5-7; 8-10; 16-18; 19-21; 26-28; 29-31; 36-38; All; including functional portions comprising all of SEQ ID NOs: 16-21; all of SEQ ID NOs: 26-31; More preferably, the polypeptide comprises a functional moiety comprising all of SEQ ID NOs: 5-10 or all amino acid sequences of SEQ ID NOs: 26-31.

  Alternatively, or in addition, a polypeptide of the invention may comprise a variable region of a TCR of the invention comprising, for example, a combination of the CDR regions described above. In this regard, the polypeptide comprises SEQ ID NO: 11, 22, 32 or 42 (α chain variable region), SEQ ID NO: 12, 23, 33 or 43 (β chain variable region), both SEQ ID NOs: 11 and 12, It may comprise the amino acid sequences of both numbers 22 and 23, both SEQ ID NOs 32 and 33, or both SEQ ID NOs 42 and 43. Preferably, the polypeptide comprises the amino acid sequence of both SEQ ID NO: 11 and 12, or both SEQ ID NO: 32 and 33.

  Alternatively, or in addition, a polypeptide of the invention may comprise the full length of one α chain or β chain of the TCRs described herein. In this regard, the polypeptide of the present invention may comprise the amino acid sequence of SEQ ID NO: 13, 14, 24, 25, 34, 35, 44, or 45. Alternatively, the polypeptides of the present invention can comprise the α and β chains of the TCRs described herein. For example, a polypeptide of the invention may comprise the amino acid sequence of both SEQ ID NOs: 13 and 14; both SEQ ID NOs: 24 and 25; both SEQ ID NOs: 34 and 35; or both SEQ ID NOs: 44 and 45. Preferably, the polypeptide comprises the amino acid sequence of both SEQ ID NO: 13 and 14, or both SEQ ID NO: 34 and 35.

  The present invention further provides proteins comprising at least one of the polypeptides described herein. “Protein” means a molecule comprising one or more polypeptide chains.

  In embodiments, the protein of the invention comprises a first polypeptide chain comprising the amino acid sequence of SEQ ID NOs: 5-7; SEQ ID NOs: 16-18; SEQ ID NOs: 26-28; or SEQ ID NOs: 36-38, and SEQ ID NO: 8-10; SEQ ID NO: 19-21; SEQ ID NO: 29-31; or a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 39-41. Alternatively or in addition, the protein of the invention comprises a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 11, 22, 32 or 42 and an amino acid sequence of SEQ ID NO: 12, 23, 33 or 43 A second polypeptide chain may be included. The protein of the present invention includes, for example, a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 13, 24, 34 or 44, and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 14, 25, 35 or 45 Can be included. In this example, the protein of the invention can be a TCR. Alternatively, for example, if the protein comprises a single polypeptide chain comprising SEQ ID NO: 13, 24, 34 or 44 and SEQ ID NO: 14, 25, 35 or 45, or the first and / or second of the protein When the polypeptide chain further comprises another amino acid sequence (for example, an amino acid sequence encoding an immunoglobulin or a portion thereof), the protein of the present invention can be a fusion protein. In this regard, the invention also provides a fusion protein comprising at least one of the polypeptides of the invention described herein, along with at least one other polypeptide. The other polypeptide may exist as a remote polypeptide of the fusion protein, or may exist as a polypeptide expressed in frame (tandem) with one of the polypeptides of the invention described herein. Other polypeptides include any peptidic or proteinaceous molecule, or portion thereof, including immunoglobulins, CD3, CD4, CD8, MHC molecules, CD1 molecules (eg, CD1a, CD1b, CD1c, CD1d, etc.). You can code, but you are not limited to these.

  A fusion protein can include one or more copies of a polypeptide of the invention and / or one or more copies of another polypeptide. For example, a fusion protein can comprise 1, 2, 3, 4, 5 or more copies of the polypeptides of the invention and / or other polypeptides. Suitable methods for making fusion proteins are known in the art and include, for example, recombinant methods. See, for example, Choi et al., Mol. Biotechnol. 31: 193-202 (2005).

  In some embodiments of the invention, the TCRs, polypeptides and proteins of the invention can be expressed as a single protein comprising a linker peptide that links the α and β chains. In this regard, the TCRs, polypeptides and proteins of the invention comprising SEQ ID NO: 13, 24, 34 or 44 and SEQ ID NO: 14, 25, 35 or 45 may further comprise a linker peptide comprising SEQ ID NO: 15 or 54. The linker peptide may advantageously facilitate the expression of recombinant TCR, polypeptide, and / or protein in the host cell. When the host cell expresses a construct containing the linker peptide, the linker peptide can be cleaved, resulting in separation of the α and β chains.

The protein of the present invention can be a recombinant antibody comprising at least one of the polypeptides of the present invention described herein. A “recombinant antibody” as used herein refers to a recombinant (eg, genetically modified) protein comprising at least one of the polypeptides of the invention and the polypeptide chain of the antibody, or a portion thereof. That means. An antibody polypeptide, or portion thereof, can be a heavy chain, light chain, heavy chain or light chain variable or constant region, single chain variable fragment (scFv), or antibody Fc, Fab or F (ab) ₂ 'fragment. Etc. The polypeptide chain of an antibody, or portion thereof, can exist as a discrete polypeptide of a recombinant antibody. Alternatively, the polypeptide chain of an antibody, or part thereof, can be present as a polypeptide expressed in frame (tandem) with a polypeptide of the invention. The antibody polypeptide, or portion thereof, can be any antibody or polypeptide of any antibody fragment, including any of the antibodies and antibody fragments described herein.

  The scope of the invention includes functional variants of the TCRs, polypeptides and proteins of the invention described herein. The term “functional variant” as used herein refers to a TCR, polypeptide or protein that has substantial or significant sequence identity or similarity to a parent TCR, polypeptide or protein; Such functional variants retain the biological activity of the TCR, polypeptide or protein from which they are mutated. A functional variant is, for example, a mutation of a TCR, polypeptide or protein (parent TCR, polypeptide or protein) described herein that retains the ability to specifically bind to MAGE-A3 or MAGE-A12. Includes the body. For such MAGE-A3 or MAGE-A12, the parent TCR has antigen specificity, or the parent polypeptide or protein is close to, comparable to or higher than the parent TCR, polypeptide or protein. In particular, it binds specifically. With respect to the parent TCR, polypeptide or protein, the functional variant is, for example, at least about 30%, 50%, 75%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or Beyond that, the amino acid sequence may be identical to the parent TCR, polypeptide or protein.

  A functional variant can comprise, for example, the amino acid sequence of a parent TCR, polypeptide or protein having at least one conservative amino acid substitution. Conservative amino acid substitutions are known in the art, such that an amino acid having a certain physical and / or chemical property is exchanged for another amino acid having the same chemical or physical property. Includes substitution. For example, conservative amino acid substitutions may include acidic amino acids substituted with other acidic amino acids (eg, Asp or Glu), other amino acids with non-polar side chains (eg, Ala, Gly, Val, Ile, Leu, Met, Amino acids having non-polar side chains substituted with Phe, Pro, Trp, Val, etc., basic amino acids substituted with other basic amino acids (Lys, Arg, etc.), other amino acids having polar side chains (Asn) , Cys, Gln, Ser, Thr, Tyr, etc.) and amino acids having polar side chains substituted.

  Alternatively, or in addition, a functional variant can comprise the amino acid sequence of a parent TCR, polypeptide or protein having at least one non-conservative amino acid substitution. In this case, it is preferred that the non-conservative amino acid substitution does not interfere with or inhibit the biological activity of the functional variant. Preferably, the non-conservative amino acid substitution enhances the biological activity of the functional variant so that the biological activity of the functional variant is enhanced relative to the parent TCR, polypeptide or protein.

  A TCR, polypeptide or protein can consist essentially of a specific amino acid sequence or a sequence described herein so that other components of a functional variant (eg, other amino acids) Does not substantially change the biological activity of the functional variant. In this regard, the TCR, polypeptide or protein of the invention can be, for example, substantially as SEQ ID NO: 13, 14, 24, 25, 34, 35, 44 or 45, both SEQ ID NO: 13 and 14, SEQ ID NO: 24 and It can consist of both 25, both SEQ ID NOs 34 and 35, or both amino acid sequences SEQ ID NOs 44 and 45. Also, for example, the TCR, polypeptide or protein of the present invention is substantially the same as SEQ ID NO: 11, 12, 22, 23, 32, 33, 42 or 43, both SEQ ID NO: 11 and 12, SEQ ID NO: 22 and 23, both SEQ ID NOs: 32 and 33, or both amino acid sequences of SEQ ID NOs: 42 and 43. Further, the TCR, polypeptide or protein of the present invention substantially comprises SEQ ID NO: 5, 16, 26 or 36 (α chain CDR1), SEQ ID NO: 6, 17, 27 or 37 (α chain CDR2), sequence No. 7, 18, 28 or 38 (α chain CDR3), SEQ ID NO: 8, 19, 29 or 39 (β chain CDR1), SEQ ID NO: 9, 20, 30 or 40 (β chain CDR2), SEQ ID NO: 10 , 21, 31 or 41 (beta chain CDR3), or any combination thereof (eg, SEQ ID NOs: 5-7; 8-10; 5-10; 16-18; 19-21; 16- 21; 26-28; 29-31; 26-31; 36-38; 39-41; or 36-41).

  The ability of a TCR, polypeptide or protein (or functional part or functional variant thereof) to specifically bind to their biological activity (eg, MAGE-A3 or MAGE-A12; the ability to detect cancer in a host; As long as it retains the ability to treat or prevent cancer in the host, etc., the TCR, polypeptide and protein of the present invention (including functional portions and functional variants) can be of any length, That is, it can contain any number of amino acids. For example, a polypeptide can be about 50 to about 5000 amino acids long (e.g., 50, 70, 75, 100, 125, 150, 175, 200, 300, 400, 500, 600, 700, 800, 900, 1000 or more). Greater amino acid length). In this regard, the polypeptides of the present invention also include oligopeptides.

  The TCRs, polypeptides and proteins of the present invention (including functional moieties and functional variants) can include synthetic amino acids in place of one or more naturally occurring amino acids. Such synthetic amino acids are known in the art and include, for example, aminocyclohexanecarboxylic acid, norleucine, α-amino n-decanoic acid, homoserine, S-acetylaminomethyl-cysteine, trans-3- and trans-4. -Hydroxyproline, 4-aminophenylalanine, 4-nitrophenylalanine, 4-chlorophenylalanine, 4-carboxyphenylalanine, β-phenylserine β-hydroxyphenylalanine, phenylglycine, α-naphthylalanine, cyclohexylalanine, cyclohexylglycine, indoline-2 -Carboxylic acid, 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, aminomalonic acid, aminomalonic acid monoamide, N'-benzyl-N'-methyl-lysine, N ', N' Dibenzyl-lysine, 6-hydroxylysine, ornithine, α-aminocyclopentanecarboxylic acid, α-aminocyclohexanecarboxylic acid, α-aminocycloheptanecarboxylic acid, α- (2-amino-2-norbornane) -carboxylic acid, α , Γ-diaminobutyric acid, α, γ-diaminopropionic acid, homophenylalanine, and α-tert-butylglycine.

  The TCRs, polypeptides and proteins of the invention (including functional moieties and functional variants) can be glycosylated, amidated, carboxylated, phosphorylated, esterified, N-acylated (eg, via disulfide bridges). ) It may be cyclized or converted to an acid addition salt and / or optionally dimerized or polymerized or conjugated.

  When the TCRs, polypeptides and proteins (including functional moieties and functional variants) of the present invention take the form of salts, preferably the polypeptides take the form of pharmaceutically acceptable salts. Suitable pharmaceutically acceptable acid addition salts include mineral acids (hydrochloric acid, hydrobromic acid, phosphoric acid, metaphosphoric acid, nitric acid, sulfuric acid, etc.), organic acids (tartaric acid, acetic acid, citric acid, malic acid, lactic acid, Fumaric acid, benzoic acid, glycolic acid, gluconic acid, succinic acid, for example, arylsulfonic acids such as p-toluenesulfonic acid).

The TCR, polypeptide and / or protein (including functional parts and functional variants thereof) of the present invention can be obtained by methods known in the art. Suitable methods for de novo synthesis of polypeptides and proteins are described by Chan et al., Fmoc Solid Phase Peptide Synthesis , Oxford University Press, Oxford, United Kingdom, 2005; Peptide and Protein Drug Analysis , ed. Reid, R., Marcel. Dekker, Inc., 2000; Epitope Mapping , ed. Westwood et al., Oxford University Press, Oxford, United Kingdom, 2000; and references such as US Pat. No. 5,449,752. Polypeptides and proteins can also be produced recombinantly using the nucleic acids described herein using standard recombinant methods. For example, Sambrook et al., Molecular Cloning: A Laboratory Manual , 3 ^rd ed., Cold Spring Harbor Press, Cold Spring Harbor, NY 2001; and Ausubel et al., Current Protocols in Molecular Biology , Greene Publishing Associates and John Wiley & See Sons, NY, 1994. In addition, some of the TCRs, polypeptides and proteins of the present invention (including functional portions and functional variants thereof) can be obtained from sources such as plants, bacteria, insects, mammals (eg, rats, humans, etc.). Can be separated and / or purified. Isolation and purification methods are well known in the art. Alternatively, the TCRs, polypeptides and / or proteins (including functional portions and functional variants thereof) described herein can be obtained from Simpep (Dublin, CA), Peptides Technologies Corp (Gaithers, MD). Burgh) and Multiple Peptide Systems, Inc. (San Diego, CA). In this regard, the TCRs, polypeptides and proteins of the present invention can be synthetic, recombinant, isolated and / or purified.

  The scope of the present invention includes a TCR, polypeptide or protein of the present invention (including any functional part or variant thereof), nucleic acid, recombinant expression vector, host cell, host cell population, or antibody or antigen binding thereof. Conjugates (eg, bioconjugates) that include any of the moieties are included. Conjugates and generally methods for synthesizing conjugates are known in the art (eg, Hudecz, F., Methods Mol. Biol. 298: 209-223 (2005), and Kirin et al. ., Inorg Chem. 44 (15): 5405-5415 (2005)).

  “Nucleic acid” as used herein includes “polynucleotide”, “oligonucleotide” and “nucleic acid molecule” and generally means a polymer of DNA or RNA, which may be single-stranded or double-stranded. May be a chain, may be synthesized or obtained from a natural source (eg, isolated and / or purified), may contain natural, non-natural or modified nucleotides, Instead of phosphodiesters found between the nucleotides of modified oligonucleotides, natural, non-natural or modified internucleotide linkages (such as phosphoramide linkages or phosphorothioate linkages) may be included. It is generally preferred that the nucleic acid does not contain any insertions, deletions, inversions and / or substitutions. However, as discussed herein, it may be preferred for the nucleic acid to contain one or more insertions, deletions, inversions and / or substitutions.

  Preferably, the nucleic acid of the present invention can be recombinant. The term “recombinant” as used herein refers to (i) a molecule constructed outside a living cell by linking a natural or synthetic nucleic acid segment to a nucleic acid molecule capable of replicating in the living cell, Or (ii) refers to a molecule resulting from replication of the molecule described in (i) above. For purposes herein, replication can be in vitro replication or in vivo replication.

Nucleic acids can be constructed based on chemical synthesis and / or enzymatic ligation reactions using procedures known in the art. See, for example, (above) Sambrook et al., And (above) Ausubel et al. For example, a nucleic acid is designed to increase the biological stability of a naturally occurring nucleotide or molecule, or to increase the physical stability of a duplex formed upon hybridization. Can be chemically synthesized using a variety of modified nucleotides such as phosphorothioate derivatives and acridine substituted nucleotides. Examples of modified nucleotides that can be used to produce nucleic acids include 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5- (carboxyhydroxy Methyl) uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, β-D-galactosylqueosine, inosine, N ⁶ -isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N ⁶ -substituted adenine, 7-methylguanine, 5- Me Le aminomethyl uracil, 5-methoxy aminomethyl-2-thiouracil, beta-D-mannosyl queue o Shin (β-D-mannosylqueosine), 5'- methoxy carboxymethyl uracil, 5-methoxy uracil, 2-methylthio--N ⁶ - Isopentenyl adenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, Examples include, but are not limited to, 5-methyluracil, uracil-5-oxyacetic acid methyl ester, 3- (3-amino-3-N-2-carboxypropyl) uracil and 2,6-diaminopurine. It is not. Alternatively, one or more of the nucleic acids of the invention can be purchased from companies such as Macromolecular Resources (Fort Collins, CO) and Synthegen (Houston, TX).

  The nucleic acid can comprise any nucleotide sequence that encodes any of the TCRs, polypeptides or proteins described herein, or functional parts or functional variants thereof. For example, the nucleic acid can comprise, consist of, or consist essentially of any one or more of the nucleotide sequences of SEQ ID NOs: 46-49.

  The present invention also provides stringency to a nucleotide sequence that is complementary to any nucleotide sequence of a nucleic acid described herein, or to any nucleotide sequence of a nucleic acid described herein. Nucleic acids comprising nucleotide sequences that hybridize under mild conditions are provided.

  Nucleotide sequences that hybridize under stringent conditions preferably hybridize under high stringency conditions. “High stringency conditions” are those in which a nucleotide sequence specifically hybridizes to a target sequence (any nucleotide sequence of the nucleic acids described herein) in an amount that is detectably stronger than non-specific hybridization. It means to soy. For high stringency conditions, a polynucleotide having an exactly complementary sequence, or a polynucleotide containing only a few scattered mismatches, a small region of several bases (for example, 3 to 10 bases) matching the nucleotide sequence. A condition for discriminating from a random sequence by chance is included. Such complementary subregions are more easily melted than full-length complements of 14-17 or more bases and can be easily distinguished by high stringency hybridization. Conditions of relatively high stringency include, for example, low salt and / or high temperature conditions (eg, provided at a temperature of about 50-70 ° C. by about 0.02-0.1 M NaCl or equivalent). included. Such high stringency conditions are particularly suitable for detecting the expression of any of the TCRs of the present invention, if allowed, tolerate little mismatch between the nucleotide sequence and the template or target strand. It is generally understood that conditions can be made more stringent by the addition of increasing amounts of formamide.

  The present invention also includes at least about 70% or more (eg, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 94% or more with any of the nucleic acids described herein. 95%, about 96%, about 97%, about 98% or about 99%) nucleic acids comprising nucleotide sequences that are identical are provided.

  The nucleic acid of the present invention can be incorporated into a recombinant expression vector. In this regard, the present invention provides a recombinant expression vector comprising any of the nucleic acids of the present invention. For purposes herein, the term “recombinant expression vector” means a genetically modified oligonucleotide or polynucleotide construct that allows expression of mRNA, protein, polypeptide or peptide by a host cell, where The construct comprises a nucleotide sequence encoding mRNA, protein, polypeptide or peptide, and the vector is contacted with the cell under conditions sufficient to express the mRNA, protein, polypeptide or peptide in the cell. The vector of the present invention as a whole is not naturally occurring. However, some of the vectors can be naturally occurring. The recombinant expression vectors of the present invention may be DNA and RNA (single stranded or double stranded, may be synthesized or partially derived from natural sources, and may contain natural, non-natural or modified nucleotides. Can include any type of nucleotides, including but not limited to. A recombinant expression vector may contain naturally occurring internucleotide linkages or non-naturally occurring internucleotide linkages, or both types of linkages. Preferably, non-naturally occurring or modified nucleotides or internucleotide linkages do not interfere with transcription or replication of the vector.

  The recombinant expression vector of the present invention can be any suitable recombinant expression vector and can be used to transform or transfect any suitable host. Suitable vectors include vectors designed for propagation and propagation, such as plasmids and viruses, or for expression, or both. The vectors are pUC series (Fermentas Life Sciences), pBluescript series (Stratagene, La Jolla, CA), pET series (Novagen, WI, Madison), pGEX series (Pharmacia Biotech, Uppsala, Sweden) And pEX series (Clontech, Palo Alto, CA). Bacteriophage vectors such as λGT10, λGT11, λZapII (Stratagene), λEMBL4 and λNM1149 can also be used. Examples of plant expression vectors include pBI01, pBI101.2, pBI101.3, pBI121 and pBIN19 (Clontech). Examples of animal expression vectors include pEUK-Cl, pMAM, and pMAMneo (Clontech). Preferably, the recombinant expression vector is a viral vector (eg, a retroviral vector).

  The recombinant expression vectors of the invention can be prepared, for example, using standard recombinant DNA techniques as described in (above) Sambrook et al. And (above) Ausubel et al. Circular or linear expression vector constructs can be prepared to include replication systems that function in prokaryotic or eukaryotic host cells. The replication system can be derived from, for example, ColEl, 2μ plasmid, λ, SV40, bovine papilloma virus, and the like.

  Desirably, the recombinant expression vector is specific to the type of host (eg, bacteria, fungi, plant, or animal) into which the vector is introduced, considering whether the vector is DNA-based or RNA-based, as appropriate. Transcriptional and regulatory sequences such as translation initiation and termination codons.

  The recombinant expression vector may include one or more marker genes that allow selection of transformed or transfected hosts. Marker genes include biocide resistance (eg, resistance to antibiotics, heavy metals, etc.), complementation in auxotrophic hosts to provide prototrophy. Suitable marker genes for the expression vector of the present invention include, for example, neomycin / G418 resistance gene, hygromycin resistance gene, histidinol resistance gene, tetracycline resistance gene and ampicillin resistance gene.

  The recombinant expression vector is complementary to a nucleotide sequence encoding a TCR, polypeptide or protein (including functional portions and functional variants thereof) or to a nucleotide sequence encoding a TCR, polypeptide or protein. Or a native or non-native promoter operably linked to the hybridizing nucleotide sequence. The choice of promoter (eg, strong promoter, weak promoter, inducible promoter, tissue-specific promoter, developmental-specific promoter) is within the ordinary skill of one skilled in the art. Similarly, combining nucleotide sequences with promoters is also within the ordinary skill of one of ordinary skill in the art. The promoter can be a non-viral promoter, or a viral promoter (eg, a cytomegalovirus (CMV) promoter, SV40 promoter, RSV promoter, and a promoter found in long repeats of mouse stem cell virus ends).

  The recombinant expression vectors of the invention can be designed for transient expression, stable expression, or both. Recombinant expression vectors can also be made for constitutive expression or for inducible expression. In addition, recombinant expression vectors can be made to contain a suicide gene.

The term “suicide gene” as used herein refers to a gene that causes a cell expressing the suicide gene to die. A suicide gene can be a gene that confers sensitivity to a substance (eg, a drug) on the cell in which the gene is expressed and causes the cell to die when the cell is contacted with or exposed to the substance. . Suicide genes are known in the art (eg, Suicide Gene Therapy: Methods and Reviews , Springer, Caroline J. (Cancer Research UK Center for Cancer Therapeutics at the Institute of Cancer Research, Sutton, Surrey, UK), Humana Press, 2004), including, for example, herpes simplex virus (HSV) thymidine kinase (TK) gene, cytosine deaminase, purine nucleoside phosphorylase and nitroreductase.

  Other embodiments of the present invention further provide host cells comprising any of the recombinant expression vectors described herein. The term “host cell” as used herein refers to any type of cell that can contain a recombinant expression vector of the invention. The host cell can be a eukaryotic cell (eg, a plant, animal, fungus or algae) or can be a prokaryotic cell (eg, a bacterium or protozoan). The host cell can be a cultured cell or a primary cell, ie a cell isolated directly from an organism (eg, human). Host cells can be adherent cells or suspension cells, ie cells that grow in suspension. Suitable host cells are known in the art, eg DH5αE. E. coli cells, Chinese hamster ovary cells, monkey VERO cells, COS cells, HEK293 cells and the like are included. For purposes of amplifying or replicating the recombinant expression vector, the host cell is preferably a prokaryotic cell (eg, DH5 cell). For the purpose of producing a recombinant TCR, polypeptide or protein, the host cell is preferably a mammalian cell. Most preferably, the host cell is a human cell. The host cell can be any type of cell, can be from any type of tissue and can be of any developmental stage, but the host cell is preferably peripheral blood lymphocyte (PBL) or peripheral blood It is a mononuclear cell (PBMC). More preferably, the host cell is a T cell.

For purposes herein, a T cell may be any T cell (a cultured T cell (eg, primary T cell), or a T cell from a cultured T cell line (eg, Jurkat, SupT1, etc.) or a mammalian cell. T cells obtained from animals, etc.). When obtained from a mammal, T cells can be obtained from a number of sources, including but not limited to blood, bone marrow, lymph nodes, thymus or other tissues or fluids. T cells can also be enriched or purified. Preferably, the T cell is a human T cell. More preferably, the T cell is a T cell isolated from a human. T cells can be any type of T cell and can be of any developmental stage (CD4 ⁺ / CD8 ⁺ double positive T cells, CD4 ⁺ helper T cells (eg, Th ₁ and Th ₂ cells). CD8 ⁺ T cells (eg, cytotoxic T cells), tumor infiltrating lymphocytes (TIL), memory T cells (eg, central memory effector memory cells and effector memory T cells), naive T cells, etc. Not limited to). Preferably, the T cell is a CD8 ⁺ T cell or a CD4 ⁺ T cell.

  The present invention also provides a cell population comprising at least one host cell described herein. A cell population can be at least one other cell (eg, a host cell (eg, a T cell) that does not contain any recombinant expression vector or a cell other than a T cell (eg, a B cell, macrophage, neutrophil, red blood cell, liver). In addition to cells, endothelial cells, epithelial cells, muscle cells, brain cells, etc.))), it can be a heterogeneous population comprising host cells containing any of the recombinant expression vectors described. Alternatively, the cell population can be a substantially homogeneous population, such a population comprising mainly host cells comprising a recombinant expression vector (eg consisting essentially of host cells comprising a recombinant expression vector). A population can also be a clonal population of cells, and all cells of such a population are clones of a single host cell that contains a recombinant expression vector so that all cells of the population contain the recombinant expression vector. In one embodiment of the invention, the cell population is a clonal population comprising host cells comprising a recombinant expression vector described herein.

  The invention further provides an antibody or antigen-binding portion thereof that specifically binds to any functional portion of a TCR described herein. Preferably, the functional moiety specifically binds to a cancer antigen, eg, the functional moiety is SEQ ID NO: 5, 16, 26 or 36 (α chain CDR1), 6, 17, 27 or 37 (α chain CDR2 ), 7, 18, 28 or 38 (α chain CDR3), 8, 19, 29 or 39 (β chain CDR1), 9, 20, 30 or 40 (β chain CDR2), 10, 21, 31 or 41 (β chain CDR3), SEQ ID NO: 11, 22, 32 or 42 (α chain variable region), SEQ ID NO: 12, 23, 33 or 43 (β chain variable region) amino acid sequence, or a combination thereof ( For example, 5-7; 8-10; 5-10; 16-18, 19-21; 16-21; 26-28; 29-31; 26-31; 36-38; 39-41; )including. More preferably, the functional part comprises the amino acid sequence of SEQ ID NO: 5-10 or SEQ ID NO: 26-31. In a preferred embodiment, the antibody, or antigen-binding portion thereof, binds to an epitope formed by a total of 6 CDRs (alpha chain CDRs 1-3, and beta chain CDRs 1-3). The antibody can be any type of immunoglobulin known in the art. For example, the antibody can be of any isotype (eg, IgA, IgD, IgE, IgG, IgM, etc.). The antibody can be monoclonal or polyclonal. The antibody can be a naturally occurring antibody, eg, an antibody isolated and / or purified from a mammal (eg, mouse, rabbit, goat, horse, chicken, hamster, human, etc.). Alternatively, the antibody can be a recombinant antibody (eg, a humanized antibody or a chimeric antibody). The antibody can be monomeric or multimeric. An antibody can also have any level of affinity or binding power for a functional portion of a TCR of the invention. Desirably, the antibody is specific for a functional portion of the TCR of the invention so that cross-reactivity with other peptides or proteins is minimized.

  Methods for testing antibodies for their ability to bind to any functional portion of the TCR of the invention are known in the art and include any antigen-antibody binding assay (eg, radioimmunoassay (RIA), ELISA, Western blot, immunization). Sedimentation and competitive inhibition assays, etc.) (see, eg, (see below) Janeway et al., And US Patent Application Publication No. 2002/0197266 A1).

Suitable methods for producing antibodies are known in the art. For example, standard hybridoma methods are described, for example, by Koehler and Milstein, Eur. J. Immunol., 5,511-519 (1976), Harlow and Lane (eds.), Antibodies: A Laboratory Manual, CSH Press (1988), and CA Janeway et al. (eds. ), Immunobiology, 5 th Ed., Garland Publishing, New York, has been described in NY (2001)). Alternatively, the EBV-hybridoma method (Haskard and Archer, J. Immunol. Methods, 74 (2), 361-67 (1984), and Roder et al., Methods Enzymol., 121, 140-67 (1986)), and Other methods are known in the art, such as bacteriophage vector expression systems (see, eg, Huse et al., Science, 246, 1275-81 (1989)). Further, methods for producing antibodies in non-human animals are described, for example, in US Pat. Nos. 5,545,806, 5,569,825 and 5,714,352, and US Patent Application Publication No. 2002 / 0197266A1. Yes.

Phage display methods can further be used to generate the antibodies of the invention. In this regard, phage libraries that encode the antigen-binding variable (V) domains of antibodies are standard molecular biology and recombinant DNA techniques (see, eg, Sambrook et al. (Eds.), Molecular Cloning, A Laboratory Manual, ^{3 rd Edition, Cold Spring Harbor Laboratory} Press, can be created using the New York (2001) reference). Phage encoding the variable region with the desired specificity are selected for specific binding to the desired antibody, and the complete or partial antibody is reconstituted to include the selected variable domain. The nucleic acid sequence encoding the reconstituted antibody is introduced into a suitable cell line, such as a myeloma cell used for hybridoma production, so that an antibody having the properties of a monoclonal antibody is secreted by the cell (eg, (See above) Janeway et al., (Above) Huse et al., And US Pat. No. 6,265,150).

  Antibodies can be produced by transgenic mice that are transgenic for specific heavy and light chain immunoglobulin genes. Such methods are known in the art and are described, for example, in US Pat. Nos. 5,545,806 and 5,569,825, and Janeway et al., (Supra).

  Methods for producing humanized antibodies are well known in the art, for example (see above) Janeway et al., US Pat. Nos. 5,225,539, 5,585,089 and 5,693,761, European Patent No. No. 0239400 B1, as well as British Patent No. 2188638. Humanized antibodies can also be obtained using antibody resurfacing techniques described, for example, in US Pat. No. 5,639,641 and Pedersen et al., J. Mol. Biol., 235, 959-973 (1994). Can be generated.

The invention also provides an antigen binding portion of any of the antibodies described herein. The antigen binding portion can be any portion having at least one antigen binding site, such as Fab, F (ab ′) ₂ , dsFv, sFv, diabodies and triabodies.

  A single chain variable region fragment (sFv) antibody fragment (consisting of a truncated Fab fragment comprising the variable (V) domain of an antibody heavy chain linked to the V domain of an antibody light chain via a synthetic peptide) It can be produced using conventional recombinant DNA technology techniques (see, eg, Janeway et al., Supra). Similarly, disulfide stabilized variable region fragments (dsFv) can be prepared by recombinant DNA technology (see, eg, Reiter et al., Protein Engineering, 7, 697-704 (1994)). However, the antibody fragments of the present invention are not limited to these exemplary antibody fragment types.

  The antibody or antigen-binding portion thereof can also be detected by a detectable label (eg, radioisotope, fluorophore (eg, fluorescein isothiocyanate (FITC), phycoerythrin (PE)), enzyme (eg, alkaline phosphatase, horseradish peroxidase) And elemental particles (eg, gold particles), etc.).

  TCRs, polypeptides, proteins of the invention (including functional portions and functional variants thereof), nucleic acids, recombinant expression vectors, host cells (including populations thereof), and antibodies (including antigen binding portions thereof) Can be isolated and / or purified. The term “isolated” as used herein means removed from its natural environment. The term “purified” as used herein means increased purity, but “purity” is a relative term and should always be interpreted as absolute purity. Not. For example, the purity may be at least about 50%, may exceed 60%, 70%, 80%, 90%, 95%, or may be 100%.

  TCRs, polypeptides, proteins (including functional portions and variants thereof), nucleic acids, recombinant expression vectors, host cells (including populations thereof), and antibodies (including antigen binding portions thereof) of the present invention all Is generically referred to as “the TCR material of the present invention” hereinafter, but can be formulated into a composition such as a pharmaceutical composition. In this regard, the invention relates to TCRs, polypeptides, proteins, functional parts, functional variants, nucleic acids, expression vectors, host cells (including populations thereof), and antibodies (including antigen binding parts thereof), and pharmaceuticals. A pharmaceutical composition comprising any of the acceptable carriers is provided. A pharmaceutical composition of the invention comprising any of the TCR materials of the invention may comprise a plurality of TCR materials (eg, polypeptides and nucleic acids) of the invention or two or more different TCRs. Alternatively, the pharmaceutical composition comprises other pharmaceutically active substances or agents such as chemotherapeutic agents (e.g., asparaginase, busulfan, carboplatin, cisplatin, daunorubicin, doxorubicin, fluorouracil, gemcitabine, hydroxyurea, methotrexate, paclitaxel, rituximab, In combination with vinblastine, vincristine, etc.).

  Preferably the carrier is a pharmaceutically acceptable carrier. For pharmaceutical compositions, the carrier can be any conventionally used carrier and is limited only by physicochemical considerations (eg, solubility, lack of reactivity to the active compound, etc.) and route of administration. The pharmaceutically acceptable carriers described herein (eg, vehicles, adjuvants, excipients, and diluents) are well known to those skilled in the art and are readily available to the public. It is preferred that the pharmaceutically acceptable carrier is a carrier that is chemically inert to the active substance and a carrier that does not have deleterious side effects or toxicity under the conditions of use.

  The choice of carrier will be determined in part by the particular inventive TCR material and by the particular method used to administer the inventive TCR material. Accordingly, there are a variety of suitable formulations of the pharmaceutical composition of the present invention. The following formulations for oral, aerosol, parenteral, subcutaneous, infusion, intramuscular, intraarterial, intrathecal and intraperitoneal administration are exemplary and not limiting in any way. Multiple routes can be used to administer the TCR material of the present invention, and in some examples, certain routes can result in a faster and more effective response than other routes.

  Topical formulations are well known to those skilled in the art. Such formulations are particularly suitable for application to the skin in the context of the present invention.

  Formulations suitable for oral administration include: (a) a liquid solution such as an effective amount of the TCR material of the present invention dissolved in a diluent (such as water, saline or orange juice); (b) capsule, sachet ( sachet), tablets, lozenges and troches (each containing a predetermined amount of active ingredient as a solid or granule); (c) a powder; (d) a suspension in a suitable liquid; and (e) a suitable emulsion. It can consist of Liquid formulations may include diluents such as water and alcohols (eg, ethanol, benzyl alcohol and polyethylene alcohol) with or without the addition of pharmaceutically acceptable surfactants. Capsule dosage forms can be of the usual hard or soft gelatin type, including, for example, surfactants, lubricants and inert fillers (such as lactose, sucrose, calcium phosphate and corn starch). Tablet dosage forms include lactose, sucrose, mannitol, corn starch, potato starch, alginic acid, microcrystalline cellulose, gum arabic, gelatin, guar gum, colloidal silicon dioxide, croscarmellose sodium, talc, magnesium stearate, calcium stearate, stearin One or more of zinc acid, stearic acid, and other excipients, colorants, diluents, buffers, disintegrants, humidifiers, preservatives, flavoring agents and other pharmacologically compatible excipients Can be included. A lozenge dosage form may contain the TCR material of the present invention in a perfume (usually sucrose and gum arabic or tragacanth), and pastilles are inert bases in addition to such excipients known in the art. The TCR material of the present invention is included in including (e.g., gelatin and glycerin, or sucrose and gum arabic), emulsions, gels and the like.

  The TCR material of the present invention may be made into an aerosol formulation administered via inhalation alone or in combination with other suitable ingredients. These aerosol formulations can be placed in a pressurized acceptable propellant (dichlorodifluoromethane, propane, nitrogen, etc.). They can also be formulated as medicaments for non-pressurized formulations, for example in nebulizers or atomizers. Such spray formulations can also be used to spray the mucosa.

  Formulations suitable for parenteral administration include aqueous or non-aqueous isotonic sterile injection solutions (antioxidants, buffers, bacteriostats, and solutes that make the formulation isotonic with the blood of the intended recipient). And aqueous and non-aqueous sterile suspensions (which may include suspending agents, solubilizers, thickeners, stabilizers and preservatives). The TCR material of the present invention can be a pharmaceutically acceptable surfactant (eg soap or detergent), suspension (eg pectin, carbomer, methylcellulose, hydroxypropylmethylcellulose or carboxymethylcellulose) or emulsifier and other pharmaceutical adjuvants. Pharmaceutical carriers (sterile liquids or liquid mixtures including water, saline, aqueous dextrose and related sugar solutions, alcohols such as ethanol or hexadecyl alcohol, glycols such as propylene glycol or polyethylene glycol, with or without the addition of , Dimethyl sulfoxide, glycerol, ketal (2,2-dimethyl-1,3-dioxolane-4-methanol, etc.), ether, poly (ethylene glycol, etc.) 400, oil, fatty acid, fatty acid ester or glycerin Or it may be administered in a diluent which is physiologically acceptable acetylated fatty acid glycerides, etc.) in.

  Oils that can be used in parenteral formulations include petroleum, animal oils, vegetable oils or synthetic oils. Specific examples of oils include peanut oil, soybean oil, sesame oil, cottonseed oil, corn oil, olive oil, petrolatum oil, and mineral oil. Fatty acids suitable for use in parenteral formulations include oleic acid, stearic acid and isostearic acid. Ethyl oleate and isopropyl myristate are examples of suitable fatty acid esters.

  Soaps suitable for use in parenteral formulations include alkali metal, ammonium and triethanolamine salts of fatty acids, and suitable detergents include (a) cationic detergents (eg, dimethyldialkylammonium halides and alkylpyridiniums). Halides), (b) anionic detergents (eg, alkyl, aryl and olefin sulfonates, alkyls, olefins, ethers and monoglyceride sulfates, and sulfosuccinates), (c) nonionic detergents (eg, aliphatic Amine oxides, fatty acid alkanolamides and polyoxyethylene polypropylene copolymers), (d) amphoteric detergents such as alkyl-β-aminopropionates and 2-alkyl-imidazoline quaternary ammonium salts, and (e) them A mixture of

  Parenteral formulations will typically contain from about 0.5% to about 25% by weight or more of the TCR material of the present invention in solution. Preservatives and buffers can be used. In order to minimize or eliminate irritation at the injection site, such compositions may include one or more nonionic surfactants having a hydrophilic-lipophilic balance (HLB) of about 12 to about 17. The amount of surfactant in such formulations will typically range from about 5% to about 15% by weight. Suitable surfactants include polyethylene glycol sorbitan fatty acid esters (such as sorbitan monooleate) and high molecular weight adducts of hydrophobic bases and ethylene oxide formed by condensation of propylene oxide and propylene glycol. Parenteral preparations can be presented in single or multiple dose sealed containers (such as ampoules and vials), freeze-dried (frozen) requiring only the addition of sterile liquid excipients (eg, water for injection) just prior to use. Can be stored in a dry) state. Extemporaneous injection solutions and suspensions can be prepared from sterile powders, granules, and tablets of the kind previously described.

  Injectable formulations are in accordance with the present invention. The requirements for effective pharmaceutical carriers for injectable compositions are well known to those skilled in the art (eg, Pharmaceuticals and Pharmacy Practice, JB Lippincott Company, Philadelphia, PA, Banker and Chalmers, eds., Pages 238-250). (1982) and ASHP Handbook on Injectable Drugs, Toissel, 4th ed., Pages 622-630 (1986)). Preferably, when administered to a cell (eg, a T cell), the cell is administered by injection.

  In addition to the pharmaceutical compositions described above, one skilled in the art will appreciate that the TCR materials of the present invention can be formulated as inclusion complexes or liposomes, such as cyclodextrin inclusion complexes.

  For purposes of the present invention, the amount or dose of the inventive TCR material administered should be sufficient to produce a therapeutic or prophylactic response over an appropriate period of time, for example, in a subject or animal. . For example, the dose of the TCR material of the present invention is sufficient to bind to a cancer antigen or to detect, treat or prevent cancer for a period of about 2 hours or more, eg, 12-24 hours or more from the time of administration. Must. In some embodiments, the time can be longer. The dose will be determined by the effectiveness of the particular TCR material of the invention and the condition of the animal (eg, human) and the weight of the animal (eg, human) to be treated.

  Many assays for determining the dose to be administered are known in the art. For the purposes of the present invention, a given dose of T cells was administered to one mammal out of a set of mammals to which target cells were lysed or each given a different dose of T cells. Which includes comparing the extent to which IFN-γ is secreted by T cells expressing the TCR, polypeptide or protein of the present invention, but using such an assay to determine the starting dose to be administered to a mammal be able to. The extent to which target cells are lysed or IFN-γ is secreted upon administration of a fixed dose can be analyzed by methods known in the art.

The dose of the inventive TCR material will also be determined by the presence, nature and extent of any adverse side effects that may accompany the administration of a particular inventive TCR material. Typically, the attending physician will consider various factors such as age, weight, general health, dietary habits, sex, the TCR material of the invention to be administered, route of administration, severity of symptoms being treated, etc. The dose of the TCR material of the present invention for treating individual patients will be determined. The dosage of the TCR material of the present invention is, by way of example, from about 0.001 to about 1000 mg or more per kg body weight of the subject to be treated per day, from about 0.01 to about 10 mg or more per kg body weight per day, Or, it can be from about 0.01 mg to about 1 mg or more per kg body weight per day, but these are not intended to limit the invention. In embodiments where the TCR material of the invention is a cell population, the number of cells administered can vary, for example, from about 1 × 10 ⁶ to about 1 × 10 ¹¹ cells or more.

  One skilled in the art will readily appreciate that the TCR materials of the invention can be modified into a number of forms to increase the therapeutic or prophylactic efficacy of the TCR materials of the present invention by modification. Will do. For example, the TCR materials of the present invention can be conjugated directly or indirectly by crosslinking to a targeting moiety. The practice of conjugating a compound (eg, a TCR material of the present invention) to a targeting moiety is known in the art. See, for example, Wadwa et al., J. Drug Targeting 3: 111 (1995) and US Pat. No. 5,087,616. The term “targeting moiety” as used herein refers to any molecule or substance that specifically recognizes and binds to a cell surface receptor, where the targeting moiety is defined by such surface receptor. Directs delivery of the TCR material of the invention to a population of expressing cells. Targeting moieties include cell surface receptors (eg, epidermal growth factor receptor (EGFR), T cell receptor (TCR), B cell receptor (BCR), CD28, platelet derived growth factor receptor (PDGF), nicotine. Such as, but not limited to, antibodies or fragments thereof, peptides, hormones, growth factors, cytokines, and any other natural or non-natural ligands that bind to a sex acetylcholine receptor (nAChR, etc.). The term “cross-linked” as used herein refers to any substance or molecule that links the TCR material of the present invention to a targeting moiety. Those skilled in the art will recognize that sites in the TCR material of the present invention that are not necessary for the function of the TCR material of the present invention are ideal sites for adhering cross-linking and / or targeting moieties (provided that And / or the targeting moiety adheres to the TCR material of the invention and functions of the TCR material of the invention (ie, the ability to bind to MAGE-A3 or MAGE-A12; or the ability to detect, treat or prevent cancer). ).

  Alternatively, the TCR material of the present invention can be modified into a depot form so that the manner in which the TCR material of the present invention is released into the body to which it is administered is controlled with respect to time and position within the body. (See, for example, U.S. Pat. No. 4,450,150). The depot form of the TCR material of the present invention can be, for example, an implantable composition comprising the TCR material of the present invention and a porous or non-porous material (eg, a polymer), the TCR material of the present invention comprising Encapsulated or diffused throughout the material and / or diffused by decomposition of the non-porous material. The depot is then implanted at the desired location within the body and the TCR material of the present invention is released from the implant at a predetermined rate.

  It is contemplated that the pharmaceutical compositions, TCRs, polypeptides, proteins, nucleic acids, recombinant expression vectors, host cells or cell populations of the present invention can be used in methods of treating or preventing cancer. Without being bound by any particular theory, it is believed that the TCR of the present invention specifically binds to MAGE-A3 MAGE-A12, so that the TCR (or related polypeptide or protein of the present invention) is Can mediate an immune response against target cells expressing MAGE-A3 or MAGE-A12. In this regard, the present invention provides a method for the treatment or prevention of cancer in a host, which method comprises any pharmaceutical composition, TCR, polypeptide or protein described herein, as described herein. Any nucleic acid or recombinant expression vector comprising a nucleic acid sequence encoding any TCR, polypeptide, protein, or any expression vector encoding any TCR, polypeptide or protein described herein. Administration of any host cell or cell population to the host in an amount effective to treat or prevent cancer in the host.

  The terms “treat” and “prevent” and their derivatives, as used herein, do not necessarily mean 100% or complete treatment or prevention. Rather, there are varying degrees of treatment or prevention that one skilled in the art will recognize as having a potential benefit or therapeutic effect. In this regard, the methods of the present invention may provide any amount of any level of treatment or prevention of cancer in the host. Furthermore, the treatment or prevention provided by the methods of the present invention can include treatment or prevention of one or more conditions or symptoms of the disease to be treated or prevented (eg, cancer). Also, for purposes herein, “prevention” can include delaying the onset of the disease or its symptoms or conditions.

  A method for detecting the presence of cancer in a host is also provided. Such a method comprises (i) preparing a sample comprising cancer cells from a TCR, polypeptide, protein, nucleic acid, recombinant expression vector, host cell, cell population or antibody or antigen-binding portion thereof of the invention as described herein. Contact with any of the above, thereby forming a complex and detecting the complex. Detection of the complex indicates the presence of cancer in the host.

  With respect to the methods of the invention for detecting cancer in a host, a sample of cancer cells is a whole cell, a lysate thereof or a fraction of a whole cell lysate (eg, nuclear or cytoplasmic fraction, total protein fraction, or nucleic acid). Sample).

  For the purposes of the detection method of the present invention, the host may be contacted in vitro or in vivo. Preferably, the contact is in vitro.

  Complex detection can also occur by any number of methods known in the art. For example, the TCRs, polypeptides, proteins, nucleic acids, recombinant expression vectors, host cells, cell populations, or antibodies or antigen-binding portions thereof of the invention described herein can be, for example, radioisotopes, fluorophores, (Eg, fluorescein isothiocyanate (FITC), phycoerythrin (PE)), enzymes (eg, alkaline phosphatase, horseradish peroxidase), and elemental particles (eg, gold particles).

  For purposes of the methods of the invention in which a host cell or cell population is administered, the cell can be allogeneic or autologous to the host. Preferably, the cell is a cell that is autologous to the host.

  For the methods of the present invention, cancers include sarcomas (eg, synovial sarcoma, osteosarcoma, uterine leiomyosarcoma and alveolar rhabdomyosarcoma), lymphomas (eg, Hodgkin lymphoma and non-Hodgkin lymphoma), hepatocellular carcinoma, Glioma, head cancer (eg, squamous cell carcinoma), cervical cancer (eg, squamous cell carcinoma), acute lymphocyte cancer, leukemia (eg, acute myeloid leukemia and chronic lymphocytic leukemia), bone Sarcoma, brain tumor, breast cancer, cancer of the anus, anal canal or anorectal, cancer of the eye, cancer of the intrahepatic bile duct, cancer of the joint, cancer of the neck, gallbladder or pleura, cancer of the nose, nasal cavity or middle ear, oral cavity Cancer, vulvar cancer, chronic bone marrow cancer, colon cancer (eg, colon cancer), esophageal cancer, cervical cancer, gastric cancer, gastrointestinal carcinoid tumor, hypopharyngeal cancer, laryngeal cancer, liver cancer (eg, hepatocellular carcinoma), Lung cancer (eg, non-small cell lung cancer), malignant mesothelioma, melanoma, multiple myeloma, nasopharyngeal cancer, ovarian cancer, pancreatic cancer Peritoneal, omental and mesenteric cancer, pharyngeal cancer, prostate cancer, rectal cancer, kidney cancer (eg, renal cell cancer), small intestine cancer, soft tissue cancer, gastric cancer, testicular cancer, thyroid cancer, and urothelial cancer (eg , Ureteral cancer and bladder cancer).

  In the method of the present invention, the host may be any host. Preferably the host is a mammal. The term “mammal” as used herein refers to any mammal including, but not limited to, mammals such as mice and hamsters, murine mammals, and rabbits such as rabbits. Preferably, the mammal is from a feline, including a feline (cat) and a canine (dog). It is more preferable that the mammal is a bovine including a bovine animal (cow) and a boar animal (pig), or a horse (Perssodactyla) including an equine animal (horse). Most preferably, the mammal is a primate, Ceboids or Simoids (monkey) or Anthropoids (human and ape). A particularly preferred mammal is a human.

  The following examples further illustrate the invention but, of course, should not be construed as limiting its scope.

Example 1
This example demonstrates the cloning of the TCR gene from T cell clones and the generation of TCR constructs.

Four T cell clones that recognize epitopes of the MAGE-A gene family in association with the dominant class I alleles HLA-A ^* 01 and C ^* 07 were first identified. Approximately 30% of melanoma patients population express ^HLA-A * 01, people over 95% of the ^HLA-A * 01 ⁺ people, express the ^HLA-A * 0101 subtype, 50 melanoma patients More than% patients express one of the two dominant HLA-C ^* 07 subtypes, C ^* 07 ^: 01 and C ^* 07 ^: 02.

The expressed TCR α and β chains are the two clones that recognized residues 168-176 of the protein MAGE-A3 (MAGE-A3: 168-176) in association with HLA-A ^* 01, A10 and 13-18 Isolated from. In addition, an HLA-C ^* 07 restricted TCR that recognizes a peptide corresponding to residues 170-178 of the MAGE-A12 protein (MAGE-A12: 170-178) was isolated from clone 502 and FM8.

Two MAGE-A12 reactive, HLA-C ^* 07 reactive T cell clones (PHIN LB831-501D / 19, referred to as “502” (Heidecker et al., J. Immunol., 164: 6041-6045 (2000)) and “FM8” (Panelli et al., J. Immunol., 164: 4382-4392 (2000))) and two MAGE-A3 reactive, HLA-A ^* 01 restricted T cells Clone (LAU147 CTL1 or 810 / A10, referred to as “A10” (Parmentier et al., Nat. Immunol., 11: 449-454 (2010)) and NW1000 AVP-1 13-18, referred to as “13-18” ) And α and β chains encoding functional TCRs were isolated. Briefly, oligo-dT was used to reverse transcribe total RNA isolated from T cell clones into cDNA using the SMART RACE cDNA amplification kit (Clontech, Mountain View, CA). The TCR α chain and β chain expressed by the T cell clone are combined with a primer 5′-CACTGTTGCTCTTGAA GTCC-3 ′ (SEQ ID NO: 55) complementary to the TCR α chain constant region and a 5 ′ complementary to the TCR β chain constant region. -CAGGCAGTAT CTGGAGTCATTGAG-3 '(SEQ ID NO: 56) was used in combination with an adapter primer from the SMART RNA synthesis kit to perform a 5'-RACE reaction. After sequencing the 5′-RACE product, the full length gene product was amplified using specific primers designed to amplify the appropriate full length TCR α and β chains. A10 TCR expresses AV12-1 / BV24-1, 13-18 expresses AV12-3 / BV15, 502 TCR expresses AV13-1 / BV25-1, FM8 expresses AV38-2 / BV4-3 Is expressed.

  For each of the four T cell clones, the transcripts encoding the paired α and β chains were inserted into the MSGV1 retroviral expression vector.

Example 2
This example expresses anti-MAGE-A3 TCR-A10 (SEQ ID NO: 13 and 14) and anti-MAGE-A3 TCR 13-18 (SEQ ID NO: 24 and 25) in response to HLA-A1 + / MAGE-A3 + cells. Demonstrate cell reactivity.

The ability of anti-CD3 stimulated T cells transduced with TCR-A10 (SEQ ID NO: 46) and TCR 13-18 (SEQ ID NO: 48) to recognize a panel of HLA-A ^* 01 + melanoma cell lines expressing MAGE-A3. Was evaluated. Untransduced (UT) cells and transduced cells were co-cultured overnight with various tumor cell lines (Tables 1A, 1B and FIG. 6A) and interferon-gamma (IFN-γ) (pg / ml) It was measured.

The results show that 6 out of the 8 HLA-A ^* 01 + / MAGE-A3 + melanoma cell lines evaluated were from T cells transduced with TCR A10 rather than from T cells transduced with TCR 13-18. This stimulated the release of higher levels of IFN-γ (FIG. 1A). After co-culturing T cells transduced with TCR and two HLA-A1 + melanoma cell lines A375 mel and 537 mel that expressed relatively low levels of MAGE-A3, lower levels of IFN-γ In response to such target cells, T cells transduced with TCR A10 released higher levels of IFN-γ than T cells transduced with TCR 13-18. Such a reaction was constrained by HLA-A1. Because 1300 mel lacking expression of HLA-A1 did not stimulate the release of IFN-γ from cells transduced with TCR A10 and TCR 13-18, but the parental 1300 mel cell line contains HLA-A ^{This is} because the cell line generated by transfecting 01 (referred to as 1300-A1) stimulated the release of IFN-γ from T cells transduced with TCR A10 and TCR 13-18. The HLA-A ^* 01 + kidney cancer cell line, 2661 RCC without MAGE-A3 expression, did not stimulate significant IFN-γ release from T cells transduced with TCR A10 and TCR 13-18. These results show that when cocultured with MAGE-A3 ⁺ / HLA-A1 ⁺ target cells, cells expressing TCR A10 release higher levels of IFN-γ than cells expressing TCR-13-18. It is proved that. These results also demonstrate that TCR A10 and TCR-13-18 are stimulated in the presence of MAGE-A3 + / HLA-A1 + target cells.

The results of a co-culture assay performed with transduced PBMC showed that T cells transduced with TCR A10 responded to HLA-A ^* 01 + / MAGE-A3 + tumor cell lines 397 mel, 2984 mel and 2556 mel, Produced to produce high levels of IFN-gamma. Cytokine levels were 5-10 times that generated from T cells transduced with TCR 13-18 (FIG. 6A). MAGE-A3 +, provided that the cell lines 562,624 and 2359 mel is ^HLA-A * 01-negative, MAGE-A3 negative, although the renal cancer cell line 2661 RCC is ^HLA-A * 01+ is, TCR A10 or 13 No significant levels of cytokines were stimulated from any of the T cells transduced with 18 (FIG. 6A).

  TCR transduction levels were assessed using quantitative PCR assays. Such an assay uses genomic DNA to specifically detect the forward primer (SEQ ID NO: 58) and reverse primer (SEQ ID NO: 59) and the MSGV1 retroviral LTR, but not the human endogenous retroviral sequence. Performed with the designed probe (SEQ ID NO: 60). The level of amplification product was estimated to contain approximately 80% transduced T cells by transducing with TCR against the NY-ESO-1: 157-165 epitope and staining with NY-ESO-1 tetramer. Normalized to a positive control sample of PBMC.

  It was found that the difference in the activity of T cells transduced with A10 or 13-18 TCR was not due to the difference in the frequency of transduction of the two TCRs, since such frequencies were found to be equivalent. Yes (FIG. 6B). In addition, T cells transduced with A10 TCR were incubated with 0.5 nM MAGE-A3 168-176 peptide at the lowest concentration (10 times lower than that required for recognition by cells transduced with 13-18 TCR). Recognizing the target cells (FIG. 6C), it was shown that the A10 TCR has a higher functional binding force than the 13-18 TCR.

Of fresh, uncultured tumor cells that stimulate transduced T cells with either TCR A10 (SEQ ID NO: 46) or DMF5 (TCR against HLA-A ^* 0201 / MART-1: 27-35 T cell epitope). The ability was evaluated. Untransduced (UT) cells and transduced cells were co-cultured with various fresh tumors and IFN-γ (pg / ml) was measured.

The results show that T cells transduced with TCR A10 recognize 4 of the 4 HLA-A ^* 01 + / MAGE-A3 + fresh tumors tested (FrTu2767, FrTu3178, FrTu2823 and FrTu3068), transduced with DMF5 T cells were shown to recognize both HLA-A ^* 0201 + / MART-1 + fresh tumor cells (FrTu2851 and FrTu3242) tested (FIG. 1B). T cells transduced with TCR A10, while did not recognize ^HLA-A * 0201 + Fresh tumor, T cells transduced with DMF5 does not recognize ^HLA-A * 01+ fresh tumors, due to TCR A10 IFN- It was shown that γ secretion is an HLA-A1 + / MAGE-A3 + specific response.

T cells transduced with TCR A10 and 13-18 recognized 5 of 6 MAGE-A3 + and HLA-A ^* 01 ⁺ fresh tumors (FrTu), FrTu3178, 2767, 2823, 2830 and 3068, None of the HLA-A ^* 01 ⁺ fresh tumor FrTu2685 without MAGE-A3 expression or three MAGE-A3 ⁺ fresh tumors, FrTu2181, 3242 and 2803 without HLA-A ^* 01 expression were recognized ( FIG. 7A; Table 1C).

Example 3
This example shows anti-MAGE-A12 TCR 502 (SEQ ID NO: 34 and 35) or anti-MAGE-A12 TCR FM8 (SEQ ID NO: 44 and 45) in response to co-culture with HLA-Cw ^* 07 + / MAGE-A12 + cells. This demonstrates the reactivity of cells expressing.

Anti-CD3-stimulated CD8 + T cells isolated from two patient PBMC samples were transduced with a control contract encoding a truncated human low affinity nerve growth factor receptor (NGFR) to produce TCR 502 (SEQ ID NO: 47 ) Or TCR FM8 (SEQ ID NO: 49) was evaluated for their ability to recognize a panel of Cw ^* 07 + melanoma cell lines expressing MAGE-A12.

  Two primers (SEQ ID NO: 61 and 62) designed to specifically amplify the MAGE-A12 gene product but not other members of the MAGE-A gene family, and a MAGE-A12 specific probe (SEQ ID NO: 63) was used to evaluate the expression of the MAGE-A12 gene product by Q-PCR. Antigen expression was determined using plasmid controls as a basis for estimating copy number and using glyceraldehyde 3-phosphate dehydrogenase (GAPDH) for normalization. Tumor cell lines and fresh tumors that expressed more than 1,000 copies of MAGE-A12 against 106 copies of GAPDH were positive for MAGE-A12 expression.

  Transduced cells were co-cultured overnight with various tumor cell lines (Table 2A; FIGS. 6D and 6F) and IFN-γ (pg / ml) was measured.

The results show that T cells transduced with TCR 502 recognize 8 of the 8 MAGE-A12 + melanoma cell lines tested, expressing either HLA-Cw ^* 0701 or 0702, but transduced with TCR FM8. It has been demonstrated that the introduced T cells only recognize melanoma cell lines expressing HLA-Cw ^* 0702 (FIGS. 2A, 2B; see also FIG. 6F). In addition, TCR 502-transduced T cells responded to HLA-Cw ^* 0702 + target SK23 mel, 1300 mel and 624 mel in response to higher levels of IFN- compared to TCR FM8-transduced T cells. γ was released (FIGS. 6D and 6F). 1011 mel cells expressing HLA-Cw ^* 0702 but lacking MAGE-A12 expression did not stimulate significant cytokine release from cells transduced with TCR 502 or TCR FM8. T cells transduced with a control contract encoding NGFR did not respond significantly to any of the targets tested. These results indicate that when co-cultured with MAGE-A12 + / HLA-Cw7 target cells, cells expressing TCR 502 release higher levels of IFN-γ than cells expressing TCR FM8, and TCR 502 Demonstrates that it recognizes MAGE-A12 in the context of either HLA-Cw0701 or HLA-Cw0702. These results also demonstrate that TCR A502 and TCR FM8 are stimulated in the presence of MAGE-A12 + cells.

While T cells transduced with TCR 502 recognized HLA-C ^* 0702 +, MAGE-A12 + tumor 624 mel and two HLA-C ^* 07: 01+, MAGE-A12 + tumor, 397 and 2359 mel, T cells transduced with FM8 recognized HLA-C ^* 07: 02+ tumor cell line 624 mel but did not recognize 397 and 2359 mel (FIG. 6D). Transduced population of T cells, ^HLA-C * 07 does not recognize the MAGE-A3 + melanoma cell lines 526,2556 or 2984 mel lacking the expression of, and lack the expression of the ^{MAGE-A12 HLA-C * 07} : The 01+ tumor cell line 2630 mel was not recognized (FIG. 6D). These differences were found not to be due to differences in the frequency of transduction of the two TCRs (measured as described in Example 2), since such frequencies were not affected by any TCR. This is because the same was found in the introduced cells (FIG. 6B). In addition, cells transduced with 502 TCR were incubated at a minimum concentration of 2.5 nM MAGE-A12: 170-178 (100-fold lower concentration required for recognition by cells transduced with FM8TCR). Recognized target cells (FIG. 6E), indicating that the 502 TCR has a higher functional binding force than the FM8TCR.

T cells transduced with MAGE-A12 reactive TCR were then evaluated for their response to enzyme digests of fresh uncultured tumor cells. T cells transduced with TCR 502 recognize FrTu3068, one of four MAGE-A12 ⁺ fresh tumors expressing HLA-C ^* 0701, and T cells transduced with TCR 502 and FM8 FrTu2181, one of two MAGE-A12 ⁺ tumors that expressed HLA-C ^* 07: 02, was recognized (FIG. 7B; Table 2B). T cell populations transduced with the TCR, ^HLA-C * 07:01 ^- and 07:02 ^- Fresh tumor 2767 or 2823, or, MAGE-A12 lacking the expression of MAGE-A12 ^- tumor 2685,3242 and 2803 None recognized.

Example 4
This example demonstrates a population that can be treated using the TCR of the present invention.

Approximately 28% of the patient population expresses HLA-A ^* 01 and approximately 54% of the patient population expresses HLA-Cw ^* 07. Two dominant subtypes of HLA-Cw ^* 07, -Cw ^* 0701 and Cw ^* 0702, are expressed in approximately 27% and approximately 31% of the patient population, respectively. FIG. 3A illustrates the cumulative percentage of the population that would be expected to be treatable by use of a TCR constrained by HLA-A1, HLA-A2 and / or HLA-Cw7 (normal white population) Based on the ratio of these alleles).

  Because approximately 30% of patients express high levels of MAGE-A3 and MAGE-A12, the use of the TCR of the present invention allows a much higher percentage of patients to receive TCR-based adoptive immunotherapy Become. 3B, 3C show NY-ESO-1 in the context of HLA-A2; MAGE-A3 in the context of HLA-A1; MAGE-A3 and MAGE-A12 in the context of HLA-A2; and / or HLA- Illustrates the cumulative patient population of human melanoma (FIG. 3B) and synovial cell sarcoma (FIG. 3C) that would be expected to be treated with a TCR that recognizes MAGE-A12 in the context of Cw7 .

Example 5
This example expresses TCR502 or TCR FM8 in response to co-culture with target cells expressing HLA-Cw ^* 0701 or HLA-Cw ^* 0702 pulsed with peptides of various proteins from the MAGE family Demonstrate cell reactivity.

Cells transduced with NGFR, TCR 502 (SEQ ID NO: 47), or TCR FM8 (SEQ ID NO: 49) were co-cultured with cells expressing HLA-Cw ^* 0701 or HLA-Cw ^* 0702. IFN-γ (pg / ml) secretion was measured.

The results show that when pulsed with MAGE-A12 (VRGHLYIL; SEQ ID NO: 4), cells transduced with TCR 502 recognize cells expressing HLA-Cw ^* 0701 or HLA-Cw ^* 0702, and MAGE-A12. It was demonstrated that when pulsed with (VRIGHLYIL; SEQ ID NO: 4), cells transduced with TCR FM8 recognize cells expressing HLA-Cw ^* 0702 (FIG. 4). Cells transduced with NGFR did not show any significant reactivity.

Example 6
This example demonstrates the specificity of anti-MAGE-A3 and anti-MAGE-A12 TCR.

After stimulation with anti-CD3 antibody, PMBC from a single donor is not transduced or anti-MAGE-A12 TCR 502 (SEQ ID NO: 47), anti-MAGE-A12 TCR FM8 (SEQ ID NO: 49), anti-MAGE -Were transduced with PMBC that were untransdiced or transduced with -A3 TCR A10 (SEQ ID NO: 46), anti-MAGE-A3 TCR 13-18 (SEQ ID NO: 48) or anti-MAGE-A3 TCR 112-120. For 13 days after stimulation, transduced T cells were incubated with the tumor targets listed in Table 3 in a standard 4 hour ⁵¹ Cr release test.

  As shown in FIGS. 5 (A) -5 (D), anti-MAGE-A12 TCR 502 specifically lyses tumor cells expressing MAGE-A12 and HLA-Cw7, and MAGE-A12 or HLA-Cw7. Tumor cells lacking the expression of did not lyse. Anti-MAGE-A3 TCR A10 specifically lysed tumor cells that expressed MAGE-A3 and HLA-A1, but did not lyse tumor cells lacking expression of MAGE-A3 or HLA-A1.

Example 7
This example demonstrates the specificity of anti-MAGE-A3 and anti-MAGE-A12 TCR.

Kidney cell line COS-7 ^monkey, one of ^{HLA-A * 01, C *} 07:01 or ^C * 07:02, in addition, MAGE-A3, A1, A2 , A4, A6, A9, A10 or Any of A12 was transiently transfected overnight. The next day, add either T cells transduced with TCR A10, 13-18 or non-transduced control cells, or T cells transduced with TCR 502, FM8 or non-transduced control cells After overnight co-culture, soluble IFN-gamma release was assessed by ELISA.

T cells transduced with MAGE-A3 reactive TCR A10 recognized HLA-A1 ⁺ target cells transfected with MAGE-A3, but differed in peptides 1 to 3 of the MAGE-A3: 170-178 epitope We did not recognize targets transfected with MAGE-A1, A2, A4, A6, A9, A10 or A12 constructs encoding (FIG. 8A). T cells transduced with either TCR 502 or FM8 recognized HLA-C ^* 07 ^: 02 ⁺ target transfected with MAGE-A12, but MAGE-A3, A1, A2, A4, A6, A9, A10 was not recognized (FIG. 8B). On the other hand, T cells transduced with TCR502 recognizes ^HLA-C * 07:01 ⁺ targets transfected with MAGE-A12, T cells transduced with FM8 did not recognize it. Other MAGE-A family members tested were not recognized (FIG. 8C).

Example 8
This example demonstrates the reactivity of transduced T cells.

  Negative selection using CD8 and CD4T lymphocyte enrichment kits (Becton Dickinson, Franklin Lakes, NJ) followed by purification using CD8 and CD4 magnetic beads (positive selection using CD8 and CD4 magnetic beads + CD) And CD4 + T cells were isolated. Isolated CD8 + and CD4 + cells were evaluated by fluorescence activated cell sorting (FACS) analysis, and contained less than 1% of contaminating CD4 + and CD8 + T cells, respectively.

TCR transduced and separated CD8 + and CD4 + T cell population responses to tumor cell targets were then evaluated. Highly purified CD4 + T cells transduced with TCR A10, containing less than 1% of contaminated CD8 + T cells, were MAGE-A3 + tumor cell line 397 mel and MAGE− stably transfected with HLA-A ^* 01. A low but significant level of IFN-gamma was released against the A3 + tumor cell line 1300A1 mel (Table 4A; FIG. 9B). CD8 + T cells transduced with TCR A10 released interferon-gamma in response to 397 mel or 1300-A1 mel (Table 4A; FIG. 9A). CD8 + T cells transduced with TCR 502 or TCR FM8 released interferon-gamma in response to 624 mel and TCR 502 released interferon-gamma in response to 397 mel (FIG. 9C and Table 4B). ).

  All references cited herein, including publications, patent applications and patents, are to the same extent as if each reference was individually and specifically indicated to be incorporated by reference. Incorporated herein by reference to the same extent as described herein in its entirety.

  For the description of the present invention (especially with regard to the following claims), the use of the terms “a” and “an” and “the” and “at least one” and similar indicating objects is specifically noted herein. Unless otherwise clearly contradicted by context, it should be construed to cover both the singular and plural. The use of the term “at least one” after the enumeration of one or more items (eg, “at least one of A and B”) is not expressly stated herein or otherwise clearly contradicted by context. It should be construed to mean one item selected from the listed items (A or B) or any combination of two or more of the listed items (A and B). The terms “comprising”, “having”, “including” and “containing” are open-ended terms (ie, including but not limited to) unless otherwise specified. ")". Unless otherwise stated herein, the description of a range of values is intended only to serve as an abbreviation for individually referring to each individual value that falls within that range. Values are incorporated herein as if they were individually described herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples or exemplary phrases (eg, “such as”) provided herein are intended only to make the present invention clearer, Unless otherwise required, no limitation is imposed on the scope of the invention. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

  Preferred embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of these preferred embodiments will become apparent to those skilled in the art after reading the above description. The inventors anticipate that those skilled in the art will use such variations as appropriate, and that the inventors have implemented the invention in a manner different from that specifically described herein. Is intended to be. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

Claims (32)

An isolated or purified T cell receptor (TCR) having antigen specificity for melanoma antigen family A (MAGE A) -3 in relation to HLA-A1, comprising:
(A) α chain complementarity determining region (CDR) 1 amino acid sequence of SEQ ID NO: 5, α chain CDR2 amino acid sequence of SEQ ID NO: 6, α chain CDR3 amino acid sequence of SEQ ID NO: 7, β chain CDR1 amino acid sequence of SEQ ID NO: 8, The β chain CDR2 amino acid sequence of SEQ ID NO: 9 and the β chain CDR3 amino acid sequence of SEQ ID NO: 10; or (b) the α chain CDR1 amino acid sequence of SEQ ID NO: 16, the α chain CDR2 amino acid sequence of SEQ ID NO: 17, the α of SEQ ID NO: 18 A TCR comprising a chain CDR3 amino acid sequence, a β chain CDR1 amino acid sequence of SEQ ID NO: 19, a β chain CDR2 amino acid sequence of SEQ ID NO: 20, and a β chain CDR3 amino acid sequence of SEQ ID NO: 21.   2. The isolated or purified TCR of claim 1 having antigen specificity for a MAGE-A3 epitope comprising EVDPIGHLY (SEQ ID NO: 2). (A) the α chain variable region amino acid sequence of SEQ ID NO: 11 and the β chain variable region amino acid sequence of SEQ ID NO: 12; or (b) the α chain variable region amino acid sequence of SEQ ID NO: 22 and the β chain variable region of SEQ ID NO: 23 2. The isolated or purified TCR of claim 1 comprising an amino acid sequence.   4. The isolated or purified TCR of claim 3, comprising the amino acid sequences of (a) both SEQ ID NOs: 13-14, or (b) both SEQ ID NOs: 24-25. An isolated or purified polypeptide comprising a portion of the TCR of any one of claims 1-4, wherein the portion specifically binds to MAGE-A3 and:
(A) α chain CDR1 amino acid sequence of SEQ ID NO: 5, α chain CDR2 amino acid sequence of SEQ ID NO: 6, α chain CDR3 amino acid sequence of SEQ ID NO: 7, β chain CDR1 amino acid sequence of SEQ ID NO: 8, β chain CDR2 of SEQ ID NO: 9 Amino acid sequence and β-chain CDR3 amino acid sequence of SEQ ID NO: 10; or (b) α-chain CDR1 amino acid sequence of SEQ ID NO: 16, α-chain CDR2 amino acid sequence of SEQ ID NO: 17, α-chain CDR3 amino acid sequence of SEQ ID NO: 18, SEQ ID NO: A polypeptide comprising 19 β chain CDR1 amino acid sequences, SEQ ID NO: 20 β chain CDR2 amino acid sequence, and SEQ ID NO: 21 β chain CDR3 amino acid sequence. The part is:
(A) the α chain variable region amino acid sequence of SEQ ID NO: 11 and the β chain variable region amino acid sequence of SEQ ID NO: 12; or (b) the α chain variable region amino acid sequence of SEQ ID NO: 22 and the β chain variable region of SEQ ID NO: 23 6. The isolated or purified polypeptide of claim 5, comprising an amino acid sequence.   The isolated or purified polypeptide of claim 6, wherein the portion comprises the amino acid sequence of (a) both SEQ ID NOs: 13-14 or (b) both SEQ ID NOs: 24-25.   An isolated or purified protein comprising at least one of the polypeptides of any one of claims 5-7. 5. An isolated or purified protein comprising a portion of the TCR of any one of claims 1-4, wherein the portion specifically binds to MAGE-A3 and:
(A) the first polypeptide chain comprising the α chain CDR1 amino acid sequence of SEQ ID NO: 5, the α chain CDR2 amino acid sequence of SEQ ID NO: 6, and the α chain CDR3 amino acid sequence of SEQ ID NO: 7, and the β chain CDR1 of SEQ ID NO: 8 A second polypeptide chain comprising an amino acid sequence, a β-chain CDR2 amino acid sequence of SEQ ID NO: 9 and a β-chain CDR3 amino acid sequence of SEQ ID NO: 10; or (b) an α-chain CDR1 amino acid sequence of SEQ ID NO: 16, an α chain CDR2 amino acid sequence, and a first polypeptide chain comprising the α chain CDR3 amino acid sequence of SEQ ID NO: 18, and a β chain CDR1 amino acid sequence of SEQ ID NO: 19, a β chain CDR2 amino acid sequence of SEQ ID NO: 20, and SEQ ID NO: 21 a second polypeptide chain comprising, protein containing β chain CDR3 amino acid sequence of. (A) a first polypeptide chain comprising the α chain variable region amino acid sequence of SEQ ID NO: 11 and a second polypeptide chain comprising the β chain variable region amino acid sequence of SEQ ID NO: 12; or (b) α of SEQ ID NO: 22 10. The isolated or purified protein of claim 9, comprising a first polypeptide chain comprising a chain variable region amino acid sequence and a second polypeptide chain comprising the β chain variable region amino acid sequence of SEQ ID NO: 23. (A) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 13 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 14; or (b) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 24 11. The isolated or purified protein of claim 10, comprising a chain and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 25.   The protein according to any one of claims 8 to 11, wherein the protein is a fusion protein.   The protein according to any one of claims 8 to 12, wherein the protein is a recombinant antibody.   An isolation comprising a nucleotide sequence encoding the TCR of any one of claims 1-4, the polypeptide of any one of claims 5-7, or the protein of any one of claims 8-13. Or purified nucleic acid.   15. The nucleic acid of claim 14, comprising the nucleotide sequence of SEQ ID NO: 46 or SEQ ID NO: 48.   An isolated or purified nucleic acid comprising a nucleotide sequence complementary to the nucleotide sequence of the nucleic acid of claim 15. 16. An isolated or purified nucleic acid comprising a nucleotide sequence that hybridizes under high stringency conditions to the nucleotide sequence of the nucleic acid of claim 15.   A recombinant expression vector comprising the nucleic acid of any one of claims 14-17.   19. An isolated host cell comprising the recombinant expression vector of claim 18.   20. The isolated host cell of claim 19, wherein the cell is peripheral blood lymphocyte (PBL).   21. The isolated host cell of claim 20, wherein the PBL is a T cell.   20. The isolated host cell of claim 19, wherein the cell is a tumor infiltrating lymphocyte (TIL).   23. A cell population comprising at least one host cell of any one of claims 19-22.   The TCR according to any one of claims 1 to 4, the polypeptide according to any one of claims 5 to 7, the protein according to any one of claims 8 to 13, and any one of claims 14 to 17. 24. A pharmaceutical composition comprising the nucleic acid of claim 18, the recombinant expression vector of claim 18, the host cell of any of claims 19-22, or the cell population of claim 23, and a pharmaceutically acceptable carrier. A method for detecting the presence of cancer in a host comprising:
(I) A TCR according to any one of claims 1 to 4, a polypeptide according to any one of claims 5 to 7, a protein according to any one of claims 8 to 13, A compound by contacting the nucleic acid of any one of claims 14 to 17, the recombinant expression vector of claim 18, the host cell of any of claims 19 to 22, or the cell population of claim 23. Forming a body, and (ii) detecting the complex,
The method wherein detection of the complex indicates the presence of cancer in the host.   Cancer is melanoma, breast cancer, leukemia, thyroid cancer, stomach cancer, pancreatic cancer, liver cancer, lung cancer, ovarian cancer, multiple myeloma, esophageal cancer, kidney cancer, head cancer, cervical cancer, prostate cancer, or urinary tract 26. The method of claim 25, wherein the method is skin cancer.   27. The method of claim 25 or 26, wherein the host cell is a cell that is autologous to the host.   27. The method of claim 25 or 26, wherein the cells of the population are autologous to the host.   The TCR of any one of claims 1-4, the polypeptide of any one of claims 5-7, the polypeptide of any of claims 8-13 for the manufacture of a medicament for treating or preventing cancer in a host. A protein according to any one of claims 14 to 17, a nucleic acid according to any one of claims 14 to 17, a recombinant expression vector according to claim 18, a host cell according to any one of claims 19 to 22, a cell population according to claim 23, Or use of the pharmaceutical composition of claim 24.   Cancer is melanoma, breast cancer, leukemia, thyroid cancer, stomach cancer, pancreatic cancer, liver cancer, lung cancer, ovarian cancer, multiple myeloma, esophageal cancer, kidney cancer, head cancer, cervical cancer, prostate cancer, or urinary tract 30. Use according to claim 29 which is a skin cancer.   31. Use according to claim 29 or 30, wherein the host cell is a cell that is autologous to the host.   31. Use according to claim 29 or 30, wherein the cells of the population are autologous to the host.

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